U.S. patent application number 12/642487 was filed with the patent office on 2010-07-08 for hydroprocessing microalgal oils.
This patent application is currently assigned to SOLAZYME, INC.. Invention is credited to ANTHONY G. DAY, SCOTT FRANKLIN.
Application Number | 20100170144 12/642487 |
Document ID | / |
Family ID | 42310777 |
Filed Date | 2010-07-08 |
United States Patent
Application |
20100170144 |
Kind Code |
A1 |
DAY; ANTHONY G. ; et
al. |
July 8, 2010 |
Hydroprocessing Microalgal Oils
Abstract
Fuels and other valuable compositions and compounds can be made
from oil extracted from microbial biomass and from oil-bearing
microbial biomass via hydroprocessing and/or other chemical
treatments, including the alkaline hydrolysis of glycerolipids and
fatty acid esters to fatty acid salts.
Inventors: |
DAY; ANTHONY G.; (SAN
FRANCISCO, CA) ; FRANKLIN; SCOTT; (SAN DIEGO,
CA) |
Correspondence
Address: |
Townsend and Townsend and Crew LLP/Solazyme, Inc.
Two Embarcadero Center, Eighth Floor
San Francisco
CA
94111-3834
US
|
Assignee: |
SOLAZYME, INC.
SOUTH SAN FRANCISCO
CA
|
Family ID: |
42310777 |
Appl. No.: |
12/642487 |
Filed: |
December 18, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12499033 |
Jul 7, 2009 |
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12642487 |
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PCT/US2009/040123 |
Apr 9, 2009 |
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12499033 |
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PCT/US2009/066142 |
Nov 30, 2009 |
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PCT/US2009/040123 |
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61074610 |
Jun 20, 2008 |
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61043620 |
Apr 9, 2008 |
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61118590 |
Nov 28, 2008 |
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61118994 |
Dec 1, 2008 |
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61174357 |
Apr 30, 2009 |
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61219525 |
Jun 23, 2009 |
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Current U.S.
Class: |
44/388 ;
585/733 |
Current CPC
Class: |
C07C 9/00 20130101; C07C
5/22 20130101; C12P 7/6418 20130101; C07C 7/00 20130101; C07C 4/06
20130101; C10L 1/02 20130101; C07C 1/22 20130101 |
Class at
Publication: |
44/388 ;
585/733 |
International
Class: |
C10L 1/19 20060101
C10L001/19; C07C 1/00 20060101 C07C001/00 |
Claims
1. A method of producing fuel comprising subjecting a triglyceride
oil comprising a lipid profile of at least 15% C:16 fatty acids, at
least 50% C18:1 fatty acids, at least 7% C18:2 fatty acids, and
less than 3% C10:0-C14:0 fatty acids to one or more chemical
reactions to generate alkanes, whereby fuel is produced.
2. The method of claim 1, wherein the triglyceride oil further
comprises a lipid profile of 1.3.+-.0.6% C14:0 fatty acids,
23.+-.6.5% C16:0 fatty acids, 1.0.+-.1.0% C16:1 fatty acids,
3.5.+-.1.5% C18:0 fatty acids, 62.+-.8% C18:1 fatty acids,
8.5.+-.4.0% C18:2 fatty acids, and 1.5.+-.1.0% C18:3 fatty
acids.
3. The method of claim 1, wherein the triglyceride oil further
comprises at least one of: i. less than 0.4 micrograms/ml total
carotenoids; ii. less than 0.001 micrograms/ml lycopene; iii. less
than 0.02 micrograms/ml beta carotene; iv. less than 0.2 milligrams
of chlorophyll per kilogram of oil; v. 0.40-0.60 milligrams of
gamma tocopherol per 100 grams of oil; vi. 3-9 mg campesterol per
100 grams of oil; and vii. less than 0.5 milligrams of total
tocotrienols per gram of oil.
4. The method of claim 1, wherein the triglyceride oil is blended
with one or more oil or fat compositions selected from the group
consisting of soy, rapeseed, canola, palm, palm kernel, coconut,
corn, waste vegetable, Chinese tallow, olive, sunflower, cotton
seed, chicken fat, beef tallow, porcine tallow, microalgae,
macroalgae, Cuphea, flax, peanut, choice white grease, lard,
Camelina stavia, mustard seed, cashew nut, oats, lupine, kenaf,
calendula, hemp, coffee, linseed (flax), hazelnut, euphorbia,
pumpkin seed, coriander, sesame, safflower, rice, tung tree, cocoa,
copra, pium poppy, castor beans, pecan, jojoba, jatropha,
macadamia, Brazil nuts, avocado, petroleum, or a distillate
fraction of any of the preceding oils before being subjected to one
or more chemical reactions.
5. The method of claim 1, wherein the one or more chemical
reactions is a chemical reaction selected from the group consisting
of transesterification, hydrogenation, hydrocracking,
deoxygenation, isomerization, interesterification, hydroxylation,
hydrolysis to yield free fatty acids, and saponification.
6. A fuel made from the hydrogenation and isomerization of the
triglyceride oil of claim 1.
7. A fuel made from the transesterification of the triglyceride oil
of claim 1.
8. The fuel of claim 6, wherein the ASTM D86 T10-T90 distilation
range is selected from the group consisting of at least 15.degree.
C., at least 20.degree. C., at least 25.degree. C., at least
30.degree. C., at least 35.degree. C., at least 40.degree. C., and
at least 55.degree. C.
9. The fuel of claim 7, wherein the ASTM D6751 A1 cold soak time is
less than 120 seconds.
10. The method of claim 1, wherein the one or more chemical
reactions comprises fluid catalytic cracking, whereby jet fuel is
produced.
11. The method of claim 1, wherein the one or more chemical
reactions comprises hydrodeoxygenation, whereby jet fuel is
produced.
12. A method of producing fuel comprising subjecting a triglyceride
oil isolated from Prototheca microorganisms to one or more chemical
reactions to generate alkanes, whereby fuel is produced.
13. The method of claim 12, wherein the triglyceride oil is blended
with one or more oil or fat compositions selected from the group
consisting of soy, rapeseed, canola, palm, palm kernel, coconut,
corn, waste vegetable, Chinese tallow, olive, sunflower, cotton
seed, chicken fat, beef tallow, porcine tallow, microalgae,
macroalgae, Cuphea, flax, peanut, choice white grease, lard,
Camelina stavia, mustard seed, cashew nut, oats, lupine, kenaf,
calendula, hemp, coffee, linseed (flax), hazelnut, euphorbia,
pumpkin seed, coriander, sesame, safflower, rice, tung tree, cocoa,
copra, pium poppy, castor beans, pecan, jojoba, jatropha,
macadamia, Brazil nuts, avocado, petroleum, or a distillate
fraction of any of the preceding oils before being subjected to one
or more chemical reactions.
14. The method of claim 12, wherein the one or more chemical
reactions is a chemical reaction selected from the group consisting
of transesterification, hydrogenation, hydrocracking,
deoxygenation, isomerization, interesterification, hydroxylation,
hydrolysis to yield free fatty acids, and saponification.
15. A fuel made from the hydrogenation and isomerization of the
triglyceride oil of claim 12.
16. A fuel made from the transesterification of the triglyceride
oil of claim 12.
17. The fuel of claim 15, wherein the ASTM D86 T10-T90 distillation
range is selected from the group consisting of at least 15.degree.
C., at least 20.degree. C., at least 25.degree. C., at least
30.degree. C., at least 35.degree. C., at least 40.degree. C., and
at least 55.degree. C.
18. The fuel of claim 16, wherein the ASTM D6751 A1 cold soak time
is less than 120 seconds.
19. The method of claim 12, wherein the one or more chemical
reactions comprises fluid catalytic cracking, whereby jet fuel is
produced.
20. The method of claim 12, wherein the one or more chemical
reactions comprises hydrodeoxygenation, whereby jet fuel is
produced.
21. A method of producing fuel comprising subjecting a triglyceride
oil, isolated from a microorganism having a 23S rRNA genomic
sequence with at least 75% nucleotide identity to one or more of
SEQ ID NOs: 3-11, to one or more chemical reactions to generate
alkanes, whereby fuel is produced.
22. The method of claim 21, wherein the triglyceride oil is blended
with one or more oil or fat compositions selected from the group
consisting of soy, rapeseed, canola, palm, palm kernel, coconut,
corn, waste vegetable, Chinese tallow, olive, sunflower, cotton
seed, chicken fat, beef tallow, porcine tallow, microalgae,
macroalgae, Cuphea, flax, peanut, choice white grease, lard,
Camelina stavia, mustard seed, cashew nut, oats, lupine, kenaf,
calendula, hemp, coffee, linseed (flax), hazelnut, euphorbia,
pumpkin seed, coriander, sesame, safflower, rice, tung tree, cocoa,
copra, pium poppy, castor beans, pecan, jojoba, jatropha,
macadamia, Brazil nuts, avocado, petroleum, or a distillate
fraction of any of the preceding oils before being subjected to one
or more chemical reactions.
23. A fuel made from the hydrogenation and isomerization of the
triglyceride oil of claim 21.
24. A fuel made from the transesterification of the triglyceride
oil of claim 21.
25. The fuel of claim 23, wherein the ASTM D86 T10-T90 distillation
range is selected from the group consisting of at least 15.degree.
C., at least 20.degree. C., at least 25.degree. C., at least
30.degree. C., at least 35.degree. C., at least 40.degree. C., and
at least 55.degree. C.
26. The fuel of claim 24, wherein the ASTM D6751 A1 cold soak time
is less than 120 seconds.
27. The method of claim 21, wherein the one or more chemical
reactions comprises fluid catalytic cracking, whereby jet fuel is
produced.
28. The method of claim 21, wherein the one or more chemical
reactions comprises hydrodeoxygenation, whereby jet fuel is
produced.
29. A method of chemically modifying a triglyceride oil, wherein
the oil: (a) has a lipid profile of 1.3.+-.0.6% C14:0 fatty acids,
23.+-.6.5% C16:0 fatty acids, 1.0.+-.1.0% C16:1 fatty acids,
3.5.+-.1.5% C18:0 fatty acids, 62.+-.8% C18:1 fatty acids,
8.5.+-.4.0% C18:2 fatty acids, and 1.5.+-.1.0% C18:3 fatty acids;
and (b) at least one of: i. less than 0.4 micrograms/ml total
carotenoids; ii. less than 0.001 micrograms/ml lycopene; iii. less
than 0.02 micrograms/ml beta carotene; iv. less than 0.2 milligrams
of chlorophyll per kilogram of oil; v. 0.40-0.60 milligrams of
gamma tocopherol per 100 grams of oil; vi. 3-9 mg campesterol per
100 grams of oil; and vii. less than 0.5 milligrams of total
tocotrienols per gram of oil, wherein the triglyceride oil
composition is chemically modified using one or more chemical
reactions selected from the group consisting of
transesterification, hydrogenation, hydrocracking, deoxygenation,
isomerization, interesterification, hydroxylation, hydrolysis to
yield free fatty acids, and saponification.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of U.S.
application Ser. No. 12/499,033, filed Jul. 7, 2009, which is a
continuation of International Application No. PCT/US2009/040123,
filed Apr. 9, 2009, which claims the benefit under 35 U.S.C. 119(e)
of U.S. Provisional Application No. 61/043,620, filed Apr. 9, 2008
and U.S. Provisional Patent Application No. 61/074,610, filed Jun.
20, 2008. The present application is also a continuation-in-part of
International Application No. PCT/US2009/066142, filed Nov. 30,
2009, which claims the benefit under 35 U.S.C. 119(e) of U.S.
Provisional Application No. 61/118,590, filed Nov. 28, 2008, U.S.
Provisional Application No. 61/118,994, filed Dec. 1, 2008, U.S.
Provisional Application No. 61/174,357, filed Apr. 30, 2009, and
U.S. Provisional Application No. 61/219,525, filed Jun. 23, 2009.
Each of the applications referenced above is incorporated by
reference herein in its entirety for all purposes.
REFERENCE TO A SEQUENCE LISTING
[0002] This application includes a sequence listing as shown in
pages 1-3, appended hereto.
FIELD OF THE INVENTION
[0003] The present invention relates to the production of oils,
soaps and fuels made from microorganisms. In particular, the
disclosure relates oil-bearing microalgae, methods of cultivating
them for the production of useful compounds, including lipids,
fatty acid esters, and alkanes, and the chemical modification of
such useful compounds for the production of soaps, chemicals and
fuels.
BACKGROUND OF THE INVENTION
[0004] Increased demand for energy by the global economy has placed
increasing pressure on the cost of fossil fuels. This, along with
increasing interest in reducing air pollution, has spurred the
development of domestic energy supplies and triggered the
development of non-petroleum fuels for internal combustion engines.
For compression ignition (diesel) engines, it has been shown that
the simple alcohol esters of fatty acids (biodiesel) are acceptable
as an alternative diesel fuel. Biodiesel has a higher oxygen
content than diesel derived from fossil fuels, and therefore
reduces emissions of particulate matter, hydrocarbons, and carbon
monoxide, while also reducing sulfur emissions due to a low sulfur
content (Sheehan, J., et al., Life Cycle Inventory of Biodiesel and
Petroleum Diesel for Use in an Urban Bus, National Renewable Energy
Laboratory, Report NREL/SR-580-24089, Golden, Colo. (1998);
Graboski, M. S., and R. L. McCormick, Prog. Energy Combust. Sci.,
24:125-164 (1998)).
[0005] Initial efforts at the production, testing, and use of
biodiesel employed refined edible vegetable oils (expelled or
recovered by solvent extraction of oilseeds) and animal fats (e.g.,
beef tallow) as feedstocks for fuel synthesis (see, e.g., Krawczyk,
T., INFORM, 7: 800-815 (1996); and Peterson, C. L., et al., Applied
Engineering in Agriculture, 13: 71-79 (1997). Further refinement of
the methods has enabled production of fatty acid methyl esters
(FAME) from cheaper, less highly refined lipid feedstocks such as
spent restaurant grease and soybean soapstock (see, e.g.,
Mittelbach, M., and P. Tritthart, J. Am. Oil Chem. Soc.,
65(7):1185-1187 (1988); Graboski, M. S., et al., The Effect of
Biodiesel Composition on Engine Emissions from a DDC Series 60
Diesel Engine, Final Report to USDOE/National Renewable Energy
Laboratory, Contract No. ACG-8-17106-02 (2000).
[0006] For decades, photoautotrophic growth of algae has been
proposed as an attractive method of manufacturing biodiesel from
algae; see A Look Back at the U.S. Department of Energy's Aquatic
Species Program: Biodiesel from Algae, NREL/TP-580-24190, John
Sheehan, Terri Dunahay, John Benemann and Paul Roessler (1998).
Many researchers believe that because sunlight is a "free"
resource, photoautotrophic growth of algae is the most desirable
method of culturing microalgae as a feedstock for biofuel
production (see, for example Chisti, Biotechnol Adv. 2007 May-June;
25(3):294-306: "heterotrophic production is not as efficient as
using photosynthetic microalgae . . . because the renewable organic
carbon sources required for growing heterotrophic microorganisms
are produced ultimately by photosynthesis, usually in crop
plants"). Other research has not only assumed that photoautotrophic
growth is the best way to grow microalgae for biofuels, but also
that there is no need to transesterify any material from microalgal
biomass before introduction into a diesel engine (see Screagg et
al., Enzyme and Microbial Technology, Vol. 33:7, 2003, Pages
884-889).
[0007] Photosynthetic growth methods have been the focus of
considerable research over the past several decades, spurred in
part by the U.S. Department of Energy's Office of Fuels
Development, which funded a program to develop renewable
transportation fuels from algae during the period spanning 1978 to
1996. The principal production design was centered around a series
of shallow outdoor sunlight-driven ponds designed as "raceways" in
which algae, water and nutrients were circulated around a circular
pond in proximity to a source of waste CO.sub.2 (e.g., a fossil
fuel powered electricity generating plant).
[0008] Transesterification of extracted/refined plant oils is
conventionally performed by reacting a triacylglycerol ("TAG") with
a lower-alkyl alcohol (e.g., methanol) in the presence of a
catalyst (e.g., a strong acid or strong base) to yield fatty acid
alkyl esters (e.g., fatty acid methyl esters or "FAME") and
glycerol.
[0009] As described above, traditional biodiesel production has
relied on extracted and/or refined oils (expelled or recovered by
solvent extraction of oilseeds) as a feedstock for the
transesterification process. Oil sources, including soy, palm,
coconut, and canola, are commonly used, and extraction is performed
by drying the plant material and pretreating the material (e.g., by
flaking) to facilitate penetration of the plant structure by a
solvent, such as hexane. Extraction of these oils for use as a
starting material contributes significantly to the cost of
traditional biodiesel production.
[0010] Similar to the solvent extraction processes utilized to
extract oils from dried plant materials, solvent extraction of oils
from microbial biomass is carried out in the presence of an organic
solvent. Solvent extraction in this context requires the use of a
solvent that is essentially immiscible in water, such as hexane, to
produce a solvent phase, in which the oil is soluble, and an
aqueous phase, which retains the largely non-lipid portion of the
biomass. Unfortunately, in an industrial scale production, the
volume of volatile, potentially carcinogenic, and flammable organic
solvent that must be used for efficient extraction creates
hazardous operating conditions having both environmental and worker
safety aspects. Moreover, the solvent extraction process generates
a substantial solvent waste stream that requires proper disposal,
thereby increasing overall production costs.
[0011] Alternatively, "solventless" extraction processes have been
reported; these employ an aqueous solvent comprising no more than
about 5% organic solvent for extracting lipids from microorganisms
for use as a feedstock in a transesterification process for the
production of biodiesel. Briefly, the "solventless" extraction
process includes contacting a lysed cell mixture with an aqueous
solvent containing no more than about 5% organic solvent (e.g.,
hexane) to produce a phase separated mixture. The mixture comprises
a heavier aqueous layer and a lighter layer comprising emulsified
lipids. The extraction process is repeatedly performed on the
lighter lipid layer until a non-emulsified lipid layer is obtained.
Unfortunately, the repeated isolation and washing of the lipid
layer makes the "solventless" process particularly laborious.
[0012] There remains a need for cheaper, more efficient methods for
extracting valuable biomolecules derived from lipids produced by
microorganisms and for converting them into valuable fuels and
other chemicals. The present invention meets this need.
[0013] Additionally, PCT Pub. No. 2008/151149 describes methods and
materials for cultivating microalgae for the production of oil and
particularly exemplifies the production of diesel fuel from oil
produced by the microalgae Chlorella protothecoides. There remains
a need for improved methods for producing oil in microalgae,
particularly for methods that produce oils with shorter chain
length and a higher degree of saturation and without pigments, with
greater yield and efficiency. The present invention meets this
need.
SUMMARY OF THE INVENTION
[0014] In a first aspect, the present invention relates to the
discovery that direct chemical modification of lipid-containing
microbial biomass can dramatically increase the efficiency and
decrease the cost of obtaining valuable materials derived from
those lipids. Thus, in a first embodiment, then invention provides
a method of chemically modifying lipid-containing microbial biomass
including the steps of culturing a population of microbes,
harvesting microbial biomass that contains at least 5% lipid by dry
cell weight (DCW), and subjecting the biomass to a chemical
reaction that covalently modifies at least 1% of the lipid. In some
embodiments, the method further includes separating the covalently
modified lipid from other components of the biomass.
[0015] In various embodiments, the ratio of the covalently modified
lipid to the biomass from which it is separated is between 10%
lipid and 90% biomass and 90% biomass and 10% lipid by dry weight.
In some embodiments, the step of separating the lipid from other
components of the biomass includes a phase separation step in which
the covalently modified lipids form a lighter non-aqueous phase and
components of the biomass form one or more heavier phases. In some
embodiments, the biomass is subjected to the chemical reaction
without a step of prior enrichment that increases the ratio of the
lipids to the non-lipid material by more than 50% by weight. In
other embodiments, the biomass is subjected to the chemical
reaction with a step of prior enrichment that increases the ratio
of the lipids to the dry weight of the microbes. In some
embodiments, the harvested biomass is not subjected to any
treatment other than the removal of water and/or lysis of the cells
before the chemical reaction. In some embodiments, the biomass
subjected to the chemical reaction contains components other than
water in the same relative proportions as the cell culture. In some
embodiments, the lipid content of the biomass is less than 90% of
the biomass subjected to the chemical reaction.
[0016] In one embodiment, chemical modification of the
lipid-containing microbial biomass comprises transesterifying the
biomass to generate a lipophilic phase containing fatty acid alkyl
esters and a hydrophilic phase containing cell material and
glycerol. In some embodiments, the method further comprises
removing water from the biomass prior to subjecting the biomass to
the transesterifying chemical reaction. In other embodiments, the
method further comprises removing water from the biomass after the
disrupting of the biomass. In some embodiments, removing water from
the biomass is performed using a method selected from the group
consisting of lyophilization, drum drying, and oven drying the
biomass.
[0017] In some embodiments, in which the chemical modification of
the lipid-containing microbial biomass comprises transesterifying
the biomass, the method further comprises disrupting the biomass
prior to transesterifying the biomass. In some embodiments, water
is removed from the biomass prior to the disrupting of the biomass.
In some embodiments, disrupting the biomass comprises heating the
biomass to generate a lysate. In other embodiments, disrupting the
biomass comprises contacting the biomass with an acid or base
sufficient to generate a lysate. In still other embodiments,
disrupting the biomass comprises contacting the biomass with one or
more enzymes to generate a lysate. In some embodiments, the biomass
is contacted with at least one protease and at least one
polysaccharide-degrading enzyme. In some embodiments, disrupting
the biomass comprises mechanically lysing the population of
microbes to generate a lysate. In other embodiments, disrupting the
biomass comprises subjecting the biomass to osmotic shock to
generate a lysate. In still other embodiments, disrupting the
biomass comprises infecting the population of microbes with a lytic
virus to generate a lysate. In other embodiments, disrupting the
biomass comprises inducing the expression of a lytic gene within
the population of microbes to promote autolysis and generation of a
lysate.
[0018] In some embodiments of the chemical modification method in
which the chemical reaction comprises transesterification, the
fatty acid alkyl esters are fatty acid methyl esters or fatty acid
ethyl esters. In some embodiments, transesterifying the biomass
comprises contacting the biomass with an alcohol and a base. In
some embodiments, the alcohol is selected from methanol, ethanol,
propanol, isopropanol, and mixtures thereof. In some embodiments,
the base is selected from NaOH, KOH, and mixtures thereof. In one
embodiment, the alcohol is methanol and the base is NaOH. In some
embodiments, transesterifying the biomass comprises contacting the
biomass with an alcohol and a lipase. In some embodiments, the
lipase is expressed from an exogenous lipase gene within the
population of microbes. In some embodiments, expression of the
exogenous lipase gene is induced by contacting the biomass with a
stimulus to activate an inducible promoter controlling expression
of the exogenous lipase gene.
[0019] In various embodiments, the amount of calcium and magnesium,
combined, by weight in the lipophilic phase is no greater than 5
parts per million. In some embodiments, the amount of phosphorous
in the lipophilic phase is no greater than 0.001%, by mass. In some
embodiments, the amount of sulfur in the lipophilic phase is no
greater than 15 parts per million. In some embodiments, the amount
of potassium and sodium, combined, by weight in the lipophilic
phase is no greater than 5 parts per million. In some embodiments,
the total carotenoid content of the lipophilic phase is no greater
than 100 micrograms of carotenoid per gram. In some embodiments,
the total chlorophyll content in the lipophilic phase is no greater
than 0.1 mg/kg.
[0020] In some embodiments, subjecting the biomass to a chemical
reaction includes contacting the biomass with an enzyme to catalyze
the chemical reaction. In some embodiments, the enzyme is a lipase.
In one embodiment, the method further comprises separating a
lipophilic phase containing the covalently modified lipids from
hydrophilic cell material of the biomass.
[0021] In some embodiments, the microbes are a species of the genus
Chlorella, and in various embodiments, the species is selected from
the group consisting of Chlorella fusca, Chlorella protothecoides,
Chlorella pyrenoidosa, Chlorella kessleri, Chlorella vulgaris,
Chlorella saccharophila, Chlorella sorokiniana and Chlorella
ellipsoidea. In one embodiment, the species is Chlorella
protothecoides. In some embodiments, the microbes is a species of
the genus Prototheca, or the species is selected from the group
consisting of Prototheca wickerhamii, Prototheca stagnora,
Prototheca portoricensis, Prototheca moriformis, and Prototheca
zopfii. In some embodiments, the microbial biomass comprises a
mixture of biomass from two distinct strains or species of microbes
that have been separately cultured. In one embodiment, at least two
of the distinct strains or species of microbes have different
glycerolipid profiles. In some embodiments, the species has a high
degree of taxonomic similarity to members of the Prototheca genus,
such as at least 95% nucleotide identity at the 23S rRNA level, as
disclosed in the examples. In some embodiments the cell is a strain
of the species Prototheca moriformis, Prototheca krugani,
Prototheca stagnora or Prototheca zopfii, and in other embodiment
the cell has a 23S rRNA sequence with at least 70, 75, 80, 85 or
95% nucleotide identity to one or more of SEQ ID NOs: 3-11.
[0022] In various embodiments of the present invention, the
harvested biomass comprises a lipid content of at least 5%, at
least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 35%, at least 40%, at least 45%, at least 50%, at least
55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, or at least 90% by DCW. In some
embodiments, at least 20% of the lipid is C18. In some embodiments,
at least 30% of the lipid is C18. In some embodiments, at least 40%
of the lipid is C18. In some embodiments, at least 50% of the lipid
is C18. In some embodiments, at least 50% of the lipid is C16 or
longer chain lengths.
[0023] In some embodiments of the present invention, the population
of microbes expresses an exogenous sucrose utilization gene. In
some embodiments, the gene is a sucrose invertase. In some
embodiments, the population of microbes expresses an exogenous
lipid pathway enzyme. In some embodiments, the lipid pathway enzyme
comprises an acyl-ACP thioesterase. In some embodiments, the
population of microbes further expresses an exogenous "naturally
co-expressed" acyl carrier protein that is co-expressed with the
acyl-ACP thioesterase. In some embodiments, the lipid pathway
enzyme has a specificity for acting on a substrate having a
specified number of carbon atoms in a chain.
[0024] In some embodiments, chemical modification of the
lipid-containing microbial biomass comprises hydrogenating the
biomass to saturate at least a subset of unsaturated bonds in the
lipid. In some embodiments, chemical modification of the
lipid-containing microbial biomass comprises interesterifying the
biomass to generate a mixture of glycerolipids having a modified
arrangement of fatty acid constituents relative to the
glycerolipids in the harvested biomass. In some embodiments,
chemical modification of the lipid-containing microbial biomass
comprises hydroxylating the biomass to generate hydroxylated
lipids. In some embodiments, at least a portion of the hydroxylated
lipids are esterified to generate estolides. In some embodiments,
chemical modification of the lipid-containing microbial biomass
comprises hydrolyzing the biomass to generate free fatty acids from
the lipid. In some embodiments, the free fatty acids are subjected
to further chemical modification. In one embodiment, chemical
modification of the lipid-containing microbial biomass comprises
deoxygenation at elevated temperature in the presence of hydrogen
and a catalyst, isomerization in the presence of hydrogen and a
catalyst, and removal of gases and naphtha compounds.
[0025] In another embodiment, chemical modification of the
lipid-containing microbial biomass comprises saponifying the
biomass to generate fatty acid salts from the lipid. In one
embodiment, the biomass is derived from a microalgae of the genus
Prototheca. In some embodiments, saponifying the biomass comprises
contacting the biomass with a base sufficient to convert at least a
portion of the glycerolipid and/or fatty acid ester components of
the lipid to fatty acid salts. In some embodiments, the base is an
alkali metal hydroxide, such as NaOH or KOH. In some embodiments,
the method further comprises contacting the biomass with a salt to
precipitate the fatty acid salts from solution. In some
embodiments, the salt comprises a water-soluble alkali metal
halide, such as NaCl or KCl.
[0026] In some embodiments, two distinct strains or species of
microbes are separately cultured, and biomass from both cultures is
mixed prior to subjecting the biomass to a chemical reaction that
modifies at least 1% of the lipid. In some embodiments, at least
two of the distinct strains of microbes have different glycerolipid
profiles.
[0027] In one aspect, the present invention is directed to a
saponification method for making a soap. In some embodiments, the
method includes culturing a population of microbes, harvesting
microbial biomass that contains at least 5% lipid by DCW, including
glycerolipids or fatty acid esters, and subjecting the biomass to
an alkaline hydrolysis reaction to produce a soap from the chemical
conversion of at least a portion of the glycerolipids or fatty acid
esters to fatty acid salts. In some embodiments, the alkaline
hydrolysis reaction includes contacting the biomass with a base and
optionally heating the biomass. In some embodiments, the base is an
alkali metal hydroxide such as NaOH or KOH. In some embodiments,
less than 100% of the glycerolipids and fatty acid esters in the
biomass are converted to fatty acid salts. In some embodiments,
less than 1% of the glycerolipids and fatty acid esters in the
biomass are converted to fatty acid salts.
[0028] In some embodiments of the saponification method, the method
further comprises substantially separating the fatty acid salts
from other components of the biomass. Some methods of the invention
further comprise boiling the separated fatty acid salts in water
and re-precipitating the fatty acid salts by introducing a salt
into the aqueous solution to produce a purified soap. In some
embodiments, the salt is a water-soluble alkali metal halide, such
as NaCl or KCl.
[0029] Some saponification methods of the invention further
comprise combining the purified soap or saponified oil composition
with one or more additives selected from the group consisting of
essential oils, fragrance oils, flavor oils, botanicals, extracts,
CO.sub.2 extracts, clays, colorants, titanium dioxide, micas,
tinting herbs, glitters, exfoliants, fruit seeds, fibers, grain
powders, nut meals, seed meals, oil beads, wax beads, herbs,
hydrosols, vitamins, milk powders, preservatives, antioxidants,
tocopherols, salts, sugars, vegetable oils, waxes, glycerin, sea
vegetables, nutritive oils, moisturizing oils, vegetable butters,
propylene glycol, parabens, honey, bees wax, aloe, polysorbate,
cornstarch, cocoa powder, coral powder, humectants, gums,
emulsifying agents, and thickeners. In one embodiment, the mixture
is packaged as a cosmetics product. In another embodiment, the
cosmetic product comprises a facial cleanser.
[0030] In some embodiments of the saponification method, the ratio
of fatty acid salts to the biomass from which they are separated is
between 10% fatty acid salts to 90% biomass and 90% fatty acid
salts to 10% biomass by dry weight. In some methods, the biomass is
subjected to the alkaline hydrolysis reaction without a step of
prior enrichment that increases a ratio of lipid to non-lipid
material in the biomass by more than 50% by weight. In some
methods, the harvested biomass is not subjected to treatments other
than lysis before the alkaline hydrolysis reaction. In other
methods, the biomass is subjected to the alkaline hydrolysis
reaction with a step of prior enrichment that increases the ratio
of lipid to non-lipid material in the biomass as compared to the
ratio at harvesting. In some embodiments, the biomass subjected to
the alkaline hydrolysis reaction contains components other than
water in the same relative proportions as the biomass at
harvesting. In some embodiments, lipid comprises no more than 90%
of the biomass subjected to the alkaline hydrolysis reaction.
[0031] In some embodiments of the saponification method, the
harvested biomass comprises a lipid content of at least 5%, at
least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 35%, at least 40%, at least 45%, at least 50%, at least
55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, or at least 90% by DCW. In some
embodiments, the lipid comprises at least 50%, at least 60%, at
least 70%, at least 80%, at least 90%, or at least 95% saturated
fatty acid constituents.
[0032] In some embodiments, the saponification method further
comprises disrupting the biomass prior to subjecting the biomass to
the alkaline hydrolysis reaction. In some embodiments, disrupting
the biomass comprises mechanically lysing the population of
microbes to generate a lysate. In some embodiments, the oil is
extracted from the biomass before saponification. In some
embodiments, the extracted oil is substantially free of color or
pigments.
[0033] In another aspect, the present invention is directed to a
composition comprising a lighter phase containing fatty acid alkyl
esters, and at least one heavier phase containing microbial
biomass.
[0034] In various embodiments of the composition, at least 20% of
the fatty acid alkyl esters are C18. In some embodiments, at least
30% of the fatty acid alkyl esters are C18. In some embodiments, at
least 40% of the fatty acid alkyl esters are C18. In some
embodiments, at least 50% of the fatty acid alkyl esters are C18.
In some embodiments, at least 50% of the fatty acid alkyl esters
are C16 or longer chain lengths. In some embodiments, at least 10%
of the fatty acid alkyl esters are C14 or shorter chain lengths. In
some embodiments, at least 20% of the fatty acid alkyl esters are
C14 or shorter chain lengths.
[0035] In another aspect, the present invention is directed to a
composition comprising a lighter phase containing completely
saturated lipids and at least one heavier phase containing
microbial biomass. In another aspect, the present invention is
directed to a composition comprising a lighter phase containing
lipids and at least one heavier phase containing microbial biomass
from more than one species or strain. In another aspect, the
present invention is directed to a composition comprising a lighter
phase containing hydroxylated lipids, and at least one heavier
phase containing microbial biomass. In another aspect, the present
invention is directed to a composition comprising a lighter phase
containing free fatty acids and at least one heavier phase
containing microbial biomass.
[0036] In another aspect, the present invention is directed to a
composition comprising saponified oil derived from the alkaline
hydrolysis of biomass produced by culturing a population of
microbes. In some embodiments, the composition further comprises at
least one and optionally more than one oil selected from the group
of oils consisting of soy, rapeseed, canola, palm, palm kernel,
coconut, corn, waste vegetable, Chinese tallow, olive, sunflower,
cotton seed, chicken fat, beef tallow, porcine tallow, microalgae,
macroalgae, Cuphea, flax, peanut, choice white grease, lard,
Camelina sativa, mustard seed cashew nut, oats, lupine, kenaf,
calendula, hemp, coffee, linseed (flax), hazelnut, euphorbia,
pumpkin seed, coriander, camellia, sesame, safflower, rice, tung
oil tree, cocoa, copra, pium poppy, castor beans, pecan, jojoba,
jatropha, macadamia, Brazil nuts, avocado, a fossil oil or a
distillate fraction thereof.
[0037] In various embodiments, the saponified oil composition can
be a solid (including a powder), or a liquid. In some embodiments,
the composition further comprises carotenoids derived from the
biomass, and/or unsaponified glycerolipids derived from the
biomass, and/or polysaccharides derived from the biomass. In some
embodiments, the saponified oil comprises at least 50% of the
composition's total mass. In some embodiments, the saponified oil
comprises at least 75% of the composition's total mass. In other
embodiments, the saponified oil comprises less than 50% of the
composition's total mass. In other embodiments, the saponified oil
comprises less than 25% of the composition's total mass. In some
embodiments, components derived from the biomass constitute at
least 50% of the composition's total mass. In some embodiments,
components derived from the biomass constitute no more than 50% of
the composition's total mass.
[0038] In another aspect, the present invention is directed to a
kit comprising a saponified oil composition as described herein and
an oral supplement. In some embodiments, the oral supplement
comprises a vitamin or an herb.
[0039] In other embodiments the triglyceride oil composition is
blended with at least one other composition selected from the group
consisting of soy, rapeseed, canola, palm, palm kernel, coconut,
corn, waste vegetable, Chinese tallow, olive, sunflower, cotton
seed, chicken fat, beef tallow, porcine tallow, microalgae,
macroalgae, Cuphea, flax, peanut, choice white grease, lard,
Camelina sativa, mustard seed cashew nut, oats, lupine, kenaf,
calendula, hemp, coffee, linseed (flax), hazelnut, euphorbia,
pumpkin seed, coriander, camellia, sesame, safflower, rice, tung
tree, cocoa, copra, pium poppy, castor beans, pecan, jojoba,
jatropha, macadamia, Brazil nuts, avocado, petroleum, or a
distillate fraction of any of the preceding oils.
[0040] Methods of the invention also include processing the
aforementioned oils of by performing one or more chemical reactions
from the list consisting of transesterification, hydrogenation,
hydrocracking, deoxygenation, isomerization, interesterification,
hydroxylation, hydrolysis to yield free fatty acids, and
saponification. The invention also includes hydrocarbon fuels made
from hydrogenation and isomerization of the aforementioned oils and
fatty acid alkyl esters made from transesterification of the
aforementioned oils. In some embodiments the hydrocarbon fuel is
made from triglyceride isolated from cells of the genus Prototheca
wherein the ASTM D86 T10-T90 distillation range is at least
25.degree. C. In other embodiments the fatty acid alkyl ester fuel
is made from triglyceride isolated from cells of the genus
Prototheca, wherein the composition has an ASTM D6751 A1 cold soak
time of less than 120 seconds.
[0041] The invention also includes methods of manufacturing a
chemical comprising performing one or more chemical reactions from
the list consisting of transesterification, hydrogenation,
hydrocracking, deoxygenation, isomerization, interesterification,
hydroxylation, hydrolysis, and saponification on a triglyceride
oil, wherein the oil has a lipid profile of at least 4% C8-C14 and
one or more of the following attributes: 0.1-0.4 micrograms/ml
total carotenoids; less than 0.02 milligrams of chlorophyll per
kilogram of oil; 0.10-0.60 milligrams of gamma tocopherol per 100
grams of oil; 0.1-0.5 milligrams of total tocotrienols per gram of
oil, 1-8 mg per 100 grams of oil of campesterol, and 10-60 mg per
100 grams of oil of stigmasterol.
[0042] In some methods the hydrolysis reaction is selected from the
group consisting of saponification, acid hydrolysis, alkaline
hydrolysis, enzymatic hydrolysis, catalytic hydrolysis, and
hot-compressed water hydrolysis, including a catalytic hydrolysis
reaction wherein the oil is split into glycerol and fatty acids. In
further methods the fatty acids undergo an amination reaction to
produce fatty nitrogen compounds or an ozonolysis reaction to
produce mono- and dibasic-acids. In some embodiments the oil
undergoes a triglyceride splitting method selected from the group
consisting of enzymatic splitting and pressure splitting. In some
methods a condensation reaction follows the hydrolysis reaction.
Other methods include performing a hydroprocessing reaction on the
oil, optionally wherein the product of the hydroprocessing reaction
undergoes a deoxygenation reaction or a condensation reaction prior
to or simultaneous with the hydroprocessing reaction. Some methods
additionally include a gas removal reaction. Additional methods
include processing the aforementioned oils by performing a
deoxygenation reaction selected from the group consisting of: a
hydrogenolysis reaction, hydrogenation, a consecutive
hydrogenation-hydrogenolysis reaction, a consecutive
hydrogenolysis-hydrogenation reaction, and a combined
hydrogenation-hydrogenolysis reaction. In some methods a
condensation reaction follows the deoxygenation reaction. Other
methods include performing an esterification reaction on the
aforementioned oils, optionally an interestification reaction or a
transesterification reaction. Other methods include performing a
hydroxylation reaction on the aforementioned oils, optionally
wherein a condensation reaction follows the hydroxylation
reaction.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] FIG. 1 shows a chromatogram of renewable diesel produced
from Prototheca triglyceride oil.
DETAILED DESCRIPTION OF THE INVENTION
[0044] The present invention arises from the discovery that
Prototheca and certain related microorganisms have unexpectedly
advantageous properties for the production of oils, fuels and other
hydrocarbon or lipid compositions economically and in large
quantities. The oils produced by these microorganisms and the
oil-bearing biomass itself can be used in the transportation fuel,
petrochemical, and/or food and cosmetic industries, among other
applications. Transesterification of lipids or the lipid-bearing
biomass yields long-chain fatty acid esters useful as biodiesel.
Other enzymatic and chemical processes can be tailored to yield
fatty acids, aldehydes, alcohols, alkanes and alkenes. In some
applications, renewable diesel, jet fuel, or other hydrocarbon
compounds are produced. In other applications, the lipid or the
lipid-bearing biomass is saponified to produce soaps.
[0045] This detailed description of the invention is divided into
sections for the convenience of the reader. Section I provides
definitions of terms used herein. Section II provides a general
description of chemical modifications of lipids and lipid-bearing
microorganisms. Section III provides microorganisms and a
description of culture conditions useful in the methods of the
invention. Section IV provides a description of transesterification
of lipids and lipid-bearing microorganisms. Section V provides a
description of producing fuels and oleochemicals with microbial
oils. Section VI provides a description of other methods of
chemical modification of lipid-bearing microorganisms. Section VII
describes saponified compositions. Section VIII discloses examples
and embodiments of the invention. The detailed description of the
invention is followed by examples that illustrate the various
aspects and embodiments of the invention.
I. DEFINITIONS
[0046] Unless defined otherwise, all technical and scientific terms
used herein have the meaning commonly understood by a person
skilled in the art to which this invention belongs. The following
references provide one of skill with a general definition of many
of the terms used in this invention: Singleton et al., Dictionary
of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge
Dictionary of Science and Technology (Walker ed., 1988); The
Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer
Verlag (1991); and Hale & Marham, The Harper Collins Dictionary
of Biology (1991). As used herein, the following terms have the
meanings ascribed to them unless specified otherwise.
[0047] "Active in microalgae," with reference to a nucleic acid,
refers to a nucleic acid that is functional in microalgae. For
example, a promoter that has been used to drive an antibiotic
resistance gene to impart antibiotic resistance to a transgenic
microalgae is active in microalgae. Examples of promoters active in
microalgae include promoters endogenous to certain algae species
and promoters found in plant viruses.
[0048] "Acyl carrier protein" or "ACP" is a protein that binds a
growing acyl chain during fatty acid synthesis as a thiol ester at
the distal thiol of the 4'-phosphopantetheine moiety and comprises
a component of the fatty acid synthase complex. The phrase
"naturally co-expressed" with reference to an acyl carrier protein
in conjunction with a fatty acyl-ACP thioesterase means that the
ACP and the thioesterase are co-expressed naturally (in nature) in
a tissue or organism from which they are derived, e.g., because the
genes encoding the two enzymes are under the control of a common
regulatory sequence or because they are expressed in response to
the same stimulus.
[0049] "Acyl-CoA molecule" or "acyl-CoA" is a molecule comprising
an acyl moiety covalently attached to coenzyme A through a thiol
ester linkage at the distal thiol of the 4'-phosphopantetheine
moiety of coenzyme A.
[0050] "Area percent" refers to the area of peaks observed using
FAME GC/FID detection methods in which every fatty acid in the
sample is converted to a fatty acid methyl ester (FAME) prior to
detection. For example, a separate peak is observed for a fatty
acid of 14 carbon atoms with no unsaturation (C14:0) compared to
any other fatty acid such as C14:1. The peak area for each class of
FAME is directly proportional to its percent composition in the
mixture and is calculated based on the sum of all peaks present in
the sample (i.e., [area under specific peak/total area of all
measured peaks].times.100). When referring to lipid profiles of oil
and cells of the invention, "at least 20% C16" means that at least
20% of the total fatty acids in the cell or in the extracted
glycerolipid composition have a chain length that includes 16
carbon atoms.
[0051] "Axenic" means a culture of an organism that is free from
contamination by other living organisms.
[0052] "Biodiesel" refers to a fatty acid ester produced from the
transesterification of lipid. The ester can be a methyl ester,
ethyl ester, or other ester depending on the components of the
transesterification reaction.
[0053] "Biomass" refers to material produced by growth and/or
propagation of cells. Biomass may contain cells and/or
intracellular contents as well as extracellular material.
Extracellular material includes, but is not limited to, compounds
secreted by a cell.
[0054] "Bioreactor" means an enclosure or partial enclosure in
which cells, e.g., microorganisms, are cultured, optionally in
suspension.
[0055] "Catalyst" refers to an agent, such as a molecule or
macromolecular complex, capable of facilitating or promoting a
chemical reaction of a reactant to a product without becoming a
part of the product. A catalyst thus increases the rate of a
reaction, after which, the catalyst may act on another reactant to
form the product. A catalyst generally lowers the overall
activation energy required for the reaction such that the reaction
proceeds more quickly or at a lower temperature and/or a reaction
equilibrium may be more quickly attained. Examples of catalysts
include enzymes, which are biological catalysts, and heat, which is
a non-biological catalyst.
[0056] "Cellulosic material" means the products of digestion of
cellulose, such as glucose, xylose, arabinose, disaccharides,
oligosaccharides, lignin, furfurals and other molecules.
[0057] "Co-culture", and variants thereof such as "co-cultivate",
refer to the presence of two or more types of cells in the same
bioreactor. The two or more types of cells may both be
microorganisms, such as microalgae, or may be a microalgal cell
cultured with a different cell type. The culture conditions may be
those that foster growth and/or propagation of the two or more cell
types or those that facilitate growth and/or propagation of one
cell type, or a subset of the cell types, of the two or more cell
types while maintaining cellular growth for the remainder.
[0058] "Cofactor" is used herein to refer to any molecule, other
than the substrate, that is required for an enzyme to carry out its
enzymatic activity.
[0059] "Complementary DNA" ("cDNA") is a DNA copy of an mRNA, which
can be obtained, for example, by reverse transcription of messenger
RNA (mRNA) or amplification (e.g., via polymerase chain reaction
("PCR")).
[0060] "Cultivated" and variants thereof refer to the intentional
fostering of growth (increases in cell size, cellular contents,
and/or cellular activity) and/or propagation (increases in cell
numbers via mitosis) of one or more cells by use of appropriate
culture conditions. The combination of both growth and propagation
may be termed proliferation. The one or more cells may be those of
a microorganism, such as microalgae. Examples of appropriate
conditions include the use of a defined medium (with known
characteristics such as pH, ionic strength, and carbon source),
specified temperature, oxygen tension, and carbon dioxide levels in
a bioreactor. The term does not refer to the growth or propagation
of microorganisms in nature or otherwise without direct human
intervention, such as natural growth of an organism that ultimately
becomes fossilized to produce geological crude oil.
[0061] "Delipidated meal" and "delipidated microbial biomass" is
microbial biomass after oil (including lipids) has been extracted
or isolated from it, either through the use of mechanical (i.e.,
exerted by an expeller press) or solvent extracted or both.
Delipidated meal has a reduced amount of oil/lipids as compared to
before the extraction or isolation of oil/lipids from the microbial
biomass but may contain some residual oil/lipid.
[0062] "Exogenous gene" refers to a nucleic acid transformed
(introduced) into a cell. A transformed cell may be referred to as
a recombinant cell, into which additional exogenous gene(s) may be
introduced. The exogenous gene may be from a different species (and
so heterologous) or from the same species (and so homologous)
relative to the cell being transformed. In the case of a homologous
gene, the introduced gene occupies a different location in the
genome of the cell relative to the endogenous copy of the gene or
is under different regulatory controls of the endogenous gene it
replaces or both. The exogenous gene may be present in more than
one copy in the cell. The exogenous gene may be maintained in a
cell as an insertion into the genome or as an episomal
molecule.
[0063] "Exogenously provided" describes a molecule provided to the
culture media of a cell culture.
[0064] "Expeller pressing" is a mechanical method for extracting
oil from raw materials such as soybeans and rapeseed. An expeller
press is a screw type machine, which presses material through a
caged barrel-like cavity. Raw materials enter one side of the press
and spent cake exits the other side while oil seeps out between the
bars in the cage and is collected. The machine uses friction and
continuous pressure from the screw drives to move and compress the
raw material. The oil seeps through small openings that do not
allow solids to pass through. As the raw material is pressed,
friction typically causes it to heat up.
[0065] "Extracted" refers to oil or lipid separated from aqueous
biomass with or without the use of solvents.
[0066] "Fatty acyl-ACP thioesterase" is an enzyme that catalyzes
the cleavage of a fatty acid from an acyl carrier protein (ACP)
during lipid synthesis.
[0067] "Fixed carbon source" means molecule(s) containing carbon,
typically organic molecules, that are present at ambient
temperature and pressure in solid or liquid form.
[0068] "Fungus," as used herein, means heterotrophic organisms
characterized by a chitinous cell wall from the kingdom of
fungi.
[0069] "Glycerolipid profile" or "lipid profile" refers to the
distribution of different carbon chain lengths and saturation
levels of glycerolipids in a particular sample of biomass. For
example, a sample could contain glycerolipids in which
approximately 60% of the glycerolipid is C18:1, 20% is C18:0, 15%
is C16:0, and 5% is C14:0. Where a carbon length is referenced
without regard to saturation, as in "C18", such reference can
include any amount of saturation; for example, microbial biomass
that contains 20% lipid as C18 can include C18:0, C18:1, C18:2,
etc., in equal or varying amounts, the sum of which constitute 20%
of the microbial biomass.
[0070] "Homogenate" means biomass that has been physically
disrupted.
[0071] "Hydrocarbon" is (a) a molecule containing only hydrogen and
carbon atoms wherein the carbon atoms are covalently linked to form
a linear, branched, cyclic, or partially cyclic backbone to which
the hydrogen atoms are attached. The molecular structure of
hydrocarbon compounds varies from the simplest, in the form of
methane (CH.sub.4), which is a constituent of natural gas, to the
very heavy and very complex, such as some molecules such as
asphaltenes found in crude oil, petroleum, and bitumens.
Hydrocarbons may be in gaseous, liquid, or solid form, or any
combination of these forms, and may have one or more double or
triple bonds between adjacent carbon atoms in the backbone.
Accordingly, the term includes linear, branched, cyclic, or
partially cyclic alkanes, alkenes, lipids, and paraffin. Examples
include propane, butane, pentane, hexane, octane, and squalene.
[0072] "Hydrogen:carbon ratio" is the ratio of hydrogen atoms to
carbon atoms in a molecule on an atom-to-atom basis. The ratio may
be used to refer to the number of carbon and hydrogen atoms in a
hydrocarbon molecule. For example, the hydrocarbon with the highest
ratio is methane CH.sub.4 (4:1).
[0073] "Hydrophobic fraction" refers to the portion, or fraction,
of a material that is more soluble in a hydrophobic phase than in
an aqueous phase. A hydrophobic fraction is substantially
immiscible with water and usually non-polar.
[0074] "Increased lipid yield" refers to an increase in the lipid
productivity of a microbial culture, which can be achieved by, for
example, increasing dry weight of cells per liter of culture,
increasing the percentage of cells that constitute lipid, or
increasing the overall amount of lipid per liter of culture volume
per unit time.
[0075] "Inducible promoter" is a promoter that mediates
transcription of an operably linked gene in response to a
particular stimulus.
[0076] "In operable linkage" refers to a functional linkage between
two nucleic acid sequences, such as a control sequence (typically a
promoter) and the linked sequence. A promoter is in operable
linkage with an exogenous gene if it can mediate transcription of
the gene.
[0077] "In situ" means "in place" or "in its original position".
For example, a culture may contain a first microorganism, such as a
microalgae, secreting a catalyst and a second microorganism
secreting a substrate, wherein the first and second microorganisms
produce the components necessary for a particular chemical reaction
to occur in situ in the co-culture without requiring further
separation or processing of the materials.
[0078] "Lipase" is an enzyme that catalyzes the hydrolysis of ester
bonds in lipid substrates. Lipases catalyze the hydrolysis of
lipids into glycerols and fatty acids, and can function to catalyze
the transesterification of TAGs to fatty acid alkyl esters.
[0079] "Lipids" are a class of molecules that are soluble in
nonpolar solvents (such as ether and chloroform) and are relatively
or completely insoluble in water. Lipid molecules have these
properties, because they consist largely of long hydrocarbon tails
which are hydrophobic in nature. Examples of lipids include fatty
acids (saturated and unsaturated); glycerides or glycerolipids
(such as monoglycerides, diglycerides, triglycerides or neutral
fats, and phosphoglycerides or glycerophospholipids); nonglycerides
(sphingolipids, sterol lipids including cholesterol and steroid
hormones, prenol lipids including terpenoids, fatty alcohols,
waxes, and polyketides); and complex lipid derivatives
(sugar-linked lipids, or glycolipids, and protein-linked lipids).
"Fats" are a subgroup of lipids called "triacylglycerides."
[0080] A "lipid pathway enzyme" is an enzyme involved in lipid
metabolism, i.e., either lipid synthesis, modification, or
degradation, and includes, without limitation, lipases, fatty
acyl-ACP thioesterases, and acyl carrier proteins.
[0081] A "limiting concentration of a nutrient" is a nutrient
concentration in a culture that limits the propagation of a
cultured organism. A "non-limiting concentration of a nutrient" is
a nutrient concentration that can support maximal propagation
during a given culture period. Thus, the number of cells produced
during a given culture period is lower in the presence of a
limiting concentration of a nutrient than when the nutrient is
non-limiting. A nutrient is said to be "in excess" in a culture
when the nutrient is present at a concentration greater than that
which supports maximal propagation.
[0082] "Lysate" refers to a solution containing the contents of
lysed cells.
[0083] "Lysing" refers to disrupting the cellular membrane and
optionally cell wall of a cell sufficient to release at least some
intracellular contents.
[0084] "Lysis" refers to the breakage of the plasma membrane and
optionally the cell wall of a biological organism sufficient to
release at least some intracellular contents, often by mechanical,
viral or osmotic mechanisms that compromise its integrity.
[0085] "Microalgae" is a eukarytotic microbial organism that
contains a chloroplast or plastid, and optionally that is capable
of performing photosynthesis, or a prokaryotic microbial organism
capable of performing photosynthesis. Microalgae include obligate
photoautotrophs, which cannot metabolize a fixed carbon source as
energy, as well as heterotrophs, which can live solely off of a
fixed carbon source. Microalgae include unicellular organisms that
separate from sister cells shortly after cell division, such as
Chlamydomonas, as well as microbes such as, for example, Volvox,
which is a simple multicellular photosynthetic microbe of two
distinct cell types. Microalgae include cells such as Chlorella,
Dunaliella, and Prototheca. Microalgae also include other microbial
photosynthetic organisms that exhibit cell-cell adhesion, such as
Agmenellum, Anabaena, and Pyrobotrys. Microalgae also include
obligate heterotrophic microorganisms that have lost the ability to
perform photosynthesis, such as certain dinoflagellate algae
species and species of the genus Prototheca.
[0086] "Microorganism" and "microbe" are used interchangeably
herein to refer to microscopic unicellular organisms.
[0087] "Oleaginous yeast," as used herein, means yeast that can
accumulate more than 10% of DCW as lipid. Oleaginous yeast includes
yeasts such as Yarrowia lipolytica, as well as engineered strains
of yeast such as Saccharomyces cerevisiae that have been engineered
to accumulate more than 10% of the DCW as lipid.
[0088] "Osmotic shock" refers to the rupture of bacterial, algal,
or other cells in a solution following a sudden reduction in
osmotic pressure. Osmotic shock is sometimes induced to release
cellular components into a solution.
[0089] "Photobioreactor" refers to a container, at least part of
which is at least partially transparent or partially open, thereby
allowing light to pass through, in which one or more microalgae
cells are cultured. Photobioreactors may be closed, as in the
instance of a polyethylene bag or Erlenmeyer flask, or may be open
to the environment, as in the instance of an outdoor pond.
[0090] A "polysaccharide-degrading enzyme" refers to an enzyme
capable of catalyzing the hydrolysis, or depolymerization, of any
polysaccharide. For example, cellulases are polysaccharide
degrading enzymes that catalyze the hydrolysis of cellulose.
[0091] "Polysaccharides" (or "glycans") are carbohydrates made up
of monosaccharides joined together by glycosidic linkages.
Cellulose is an example of a polysaccharide that makes up certain
plant cell walls. Cellulose can be depolymerized by enzymes to
yield monosaccharides such as xylose and glucose, as well as larger
disaccharides and oligosaccharides.
[0092] "Recombinant," when used with reference, e.g., to a cell, or
nucleic acid, protein, or vector, indicates that the cell, nucleic
acid, protein, or vector, has been modified by the introduction of
a heterologous nucleic acid or protein or the alteration of a
native (naturally occurring) nucleic acid or protein, or that the
cell is derived from a cell so modified. Thus, e.g., recombinant
cells express non-native genes, genes not found in the native
(non-recombinant) form of the cell, or express native genes
differently than does the non-recombinant cell, i.e., the native
gene is over-expressed, under-expressed or not expressed at all,
relative to gene expression in the non-recombinant cell.
"Recombinant nucleic acid" refers to a nucleic acid, typically
formed in vitro by the manipulation of nucleic acid, e.g., using
polymerases and endonucleases, in a form not found in nature (and
can include purified preparations of naturally occurring nucleic
acids). Thus, an isolated nucleic acid, in a linear form, or an
expression vector formed in vitro by ligating DNA molecules that
are not normally joined (for example to place two different nucleic
acids in operable linkage with one another), are recombinant. Once
a recombinant nucleic acid is introduced into a host cell or
organism, it may replicate non-recombinantly, i.e., using the in
vivo cellular machinery of the host cell; however, such nucleic
acids, produced recombinantly and subsequently replicated
non-recombinantly, are still considered recombinant. Similarly, a
"recombinant protein" is a protein made using recombinant
techniques, i.e., through the expression of a recombinant nucleic
acid.
[0093] "Renewable diesel" is a mixture of alkanes (such as C10:0,
C12:0, C14:0, C16:0 and C18:0) produced through hydrogenation and
deoxygenation of lipids.
[0094] "Saponified oil" refers to the carboxylic acid salts and
associated compounds that are created during saponification of
fatty acid esters from microbial sources. Fatty acid esters can be
derived from the triacylgylcerols (TAGs) produced by microorganims.
Compounds associated with oils from microbial sources include
carotenoids, tocopherols, tocotrienols, and other compounds of
biological origin.
[0095] "Sonication" refers to a process of disrupting biologic
materials, such as a cell, by use of sound wave energy.
[0096] "Stover" refers to the dried stalks and leaves of a crop
remaining after a grain has been harvested.
[0097] A "sucrose utilization gene" is a gene that, when expressed,
aids the ability of a cell to utilize sucrose as an energy source.
Proteins encoded by a sucrose utilization gene are referred to
herein as "sucrose utilization enzymes" and include sucrose
transporters, sucrose invertases, and hexokinases such as
glucokinases and fructokinases.
II. GENERAL
[0098] Certain microorganisms can be used to produce lipids in
large quantities for use in the transportation fuel and
petrochemical industries, among other applications. The present
invention provides methods that significantly decrease the cost and
increase the efficiency of obtaining lipids and valuable
lipid-derived compounds form microorganisms. Suitable
microorganisms for use in the methods of the invention include
microalgae, oleaginous yeast, fungi, bacteria, and cyanobacteria.
Microalgae for use in the invention are the lipid-producing
microalgae from the genera Chlorella and Prototheca. The present
invention also provides methods for the production, isolation, and
chemical modification of lipids, particularly microalgal lipids, to
produce valuable fuels and other chemicals. The present invention
also provides methods for the in situ transesterification of
triacylglycerols (TAGs) to fatty acid alkyl esters, which are
useful as biodiesel fuels and/or for other applications,
hydroprocesing of TAGs to create renewable diesel, as well as other
methods for chemical modification of the lipids in microbial
biomass.
[0099] The present invention also provides methods of making fatty
acid alkyl esters (e.g., fatty acid methyl esters (FAME)) by
culturing a population of microbes that generate at least 5% of
their DCW as lipid, such as triglycerides. In this method, the
microbial biomass is harvested from the culture and optionally
dried to remove water. Transesterification is then accomplished by
the addition of a lower-alkyl alcohol and a catalyst (e.g., NaOH)
to generate a lipophilic phase containing the fatty acid alkyl
esters and a hydrophilic phase containing hydrophilic cell
material. The lipophilic phase can be readily separated from the
hydrophilic phase.
[0100] The direct transesterification of the biomass, without an
intervening separation process step in which the lipophilic
components are extracted from the biomass prior to
transesterification, permits production of biodiesel at greatly
reduced costs, as compared to methods which employ traditional
extraction and refining steps prior to transesterification.
[0101] The methods of the present invention provide further
advantages in the generation of biodiesel via the in situ
transesterification of glycerolipids to fatty acid alkyl esters. In
particular, the microbes of the present invention can be cultured
under conditions which permit modulation of the glycerolipid
content of the cells. Surprisingly, it has been discovered that a
greater proportion of total glycerolipids can be converted to fatty
acid alkyl esters in cells which comprise increasingly higher
oil:non-oil ratios as a function of DCW. Moreover, these higher
oil:non-oil ratios also lead to another unexpected advantage: fatty
acid alkyl esters generated from cells that comprise increasingly
higher oil:non-oil ratios have a lower concentration of heteroatoms
than those produced from cells with lower oil:non-oil ratios. The
methods provided contrast markedly with current dogma in the field,
namely that photoautotrophic growth of microalgae is the best
method of microalgae cultivation for biofuel production (see for
example, Rodolfi, et al., Biotechnology & Bioengineering
102(1):100-112 (2008) for discussion on screening microalgal
strains for their biomass productivity and lipid content for growth
in an outdoor photobioreactor). It was also discovered that the
higher the oil content of the biomass, the higher quality of the
resulting product after direct chemical modification. The present
invention provides heterotrophic methods of culturing microbes
(e.g., microalgae) to achieve higher oil content for direct
chemical modification for the production of higher quality chemical
products.
[0102] The present invention also provides other methods of
chemically modifying lipid-containing microbial biomass, including
without limitation, hydrogenation, interesterification,
hydroxylation, hydrolysis, and saponification. These methods can be
used with the various microorganisms and culturing conditions set
forth herein to produce a wide variety of chemical products for a
multitude of applications.
[0103] The present invention also provides useful compositions,
including: a composition comprising a lighter phase containing
fatty acid alkyl esters and at least one heavier phase containing
microbial biomass; a composition comprising a lighter phase
containing completely saturated lipids and at least one heavier
phase containing microbial biomass; a composition comprising a
lighter phase containing lipids and at least one heavier phase
containing microbial biomass from more than one species or strain;
a composition comprising a lighter phase containing hydroxylated
lipids and at least one heavier phase containing microbial biomass;
and a composition comprising a lighter phase containing free fatty
acids and at least one heavier phase containing microbial biomass.
The present invention also provides compositions comprising
saponified oil derived from the alkaline hydrolysis of biomass
produced by culturing a population of microorganisms.
III. MICROORGANISMS AND CULTIVATION OF MICROORGANISMS
[0104] Microorganisms useful in the invention produce lipids
suitable for chemical modification for biodiesel production and for
production of fatty acid esters for other purposes such as
industrial chemical feedstocks and edible oils, as well as the
production of other chemical entities. Suitable lipids for
biodiesel and chemicals production include TAGs containing fatty
acid molecules. In some embodiments, suitable fatty acids contain
at least 8, at least 10, at least 12, at least 14, at least 16, at
least 18, at least 20, at least 22, at least 24, at least 26, at
least 28, at least 30, at least 32, or at least 34 carbon atoms or
more. Preferred fatty acids for biodiesel generally contain 16 and
18 carbon atoms. In certain embodiments, the above fatty acids are
saturated (with no carbon-carbon double or triple bonds); mono
unsaturated (single double bond); polyunsaturated (two or more
double bonds); are linear (not cyclic); and/or have little or no
branching in their structures.
[0105] In some embodiments, culturing microorganisms useful in the
in situ transesterification and modification methods of the present
invention yields a biomass that, when dry, comprises an oil content
of at least 5%, at least 10%, at least 15%, at least 20%, or at
least 25%. In other embodiments, the dried biomass comprises an oil
content of at least 30%, at least 35%, at least 40%, at least 45%,
at least 50%, at least 55%, at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, or at least 90%.
"Dry" or "dried," as used in this context, refers to the absence of
substantially all water. Biomass can also be chemically modified
without being dried; for example, biomass includes a centrifuged
cell paste.
[0106] In some embodiments, culturing microorganisms useful in the
in situ transesterification and other chemical modification methods
of the invention yields a biomass in which at least 10% of the
lipid is C18, at least 15% of the lipid is C18, at least 20% of the
lipid is C18, or at least 25% of the lipid is C18. In other
embodiments, the biomass comprises a lipid constituent which is at
least 30% C18, at least 35% C18, at least 40% C18, at least 45%
C18, or at least 50% C18. In still other embodiments, the biomass
can comprise a lipid component that is at least 10%, at least 15%,
at least 20%, at least 25%, at least 30%, at least 35%, at least
40%, at least 45%, or at least 50% C14 and/or C16, or longer chain
lengths. Alternatively, the biomass can comprise a lipid component
that is at least 10% or at least 20% C14, or shorter chain
lengths.
[0107] A preferred microorganism useful to produce lipids for use
in the invention are microalgae in the genus Prototheca. For the
convenience of the reader, this section is subdivided into
subsections. Subsection 1 describes Prototheca species and strains
and how to identify new Prototheca species and strains related
microalgae by genomic DNA comparison. Subsection 2 describes
bioreactors useful for cultivation. Subsection 3 describes media
for cultivation. Subsection 4 describes oil/lipid production in
accordance with illustrative cultivation methods of the
invention.
[0108] A. Prototheca Species and Strains
[0109] Prototheca is a remarkable microorganism for use in the
production of lipid, because it can produce high levels of lipid,
particularly lipid suitable for fuel production. The lipid produced
by Prototheca has hydrocarbon chains of shorter chain length and a
higher degree of saturation than that produced by other microalgae.
Chain length and saturated lipid produced by Prototheca species is
suitable for saponification and other chemical modifications used
to produced chemicals and fuels. Moreover, Prototheca lipid is
generally free of pigment (low to undetectable levels of
chlorophyll and certain carotenoids) and in any event contains much
less pigment than lipid from other microalgae. Illustrative
Prototheca strains for use in the methods of the invention include
Prototheca wickerhamii, Prototheca stagnora (including UTEX 327),
Prototheca portoricensis, Prototheca moriformis (including UTEX
strains 1441, 1435), and Prototheca zopfii. Species of the genus
Prototheca are obligate heterotrophs.
[0110] Species of Prototheca for use in the invention can be
identified by amplification of certain target regions of the
genome. For example, identification of a specific Prototheca
species or strain can be achieved through amplification and
sequencing of nuclear and/or chloroplast DNA using primers and
methodology using any region of the genome, for example using the
methods described in Wu et al., Bot. Bull. Acad. Sin. (2001)
42:115-121 Identification of Chlorella spp. isolates using
ribosomal DNA sequences. Well established methods of phylogenetic
analysis, such as amplification and sequencing of ribosomal
internal transcribed spacer (ITS1 and ITS2 rDNA), 23S rRNA, 18S
rRNA, and other conserved genomic regions can be used by those
skilled in the art to identify species of not only Prototheca, but
other hydrocarbon and lipid producing organisms with similar lipid
profiles and production capability. For examples of methods of
identification and classification of algae also see for example
Genetics, 2005 August; 170(4):1601-10 and RNA, 2005 April;
11(4):361-4.
[0111] Thus, genomic DNA comparison can be used to identify
suitable species of microalgae to be used in the present invention.
Regions of conserved genomic DNA, such as but not limited to DNA
encoding for 23S rRNA, can be amplified from microalgal species and
compared to consensus sequences in order to screen for microalgal
species that are taxonomically related to the preferred microalgae
used in the present invention. Examples of such DNA sequence
comparison for species within the Prototheca genus are shown below.
Genomic DNA comparison can also be useful to identify microalgal
species that have been misidentified in a strain collection. Often
a strain collection will identify species of microalgae based on
phenotypic and morphological characteristics. The use of these
characteristics may lead to miscategorization of the species or the
genus of a microalgae. The use of genomic DNA comparison can be a
better method of categorizing microalgae species based on their
phylogenetic relationship.
[0112] Microalgae for use in the present invention typically have
genomic DNA sequences encoding for 23S rRNA that have at least 99%,
least 95%, at least 90%, or at least 85% nucleotide identity to at
least one of the sequences listed in SEQ ID NOs: 3-11.
[0113] For sequence comparison to determine percent nucleotide or
amino acid identity, typically one sequence acts as a reference
sequence, to which test sequences are compared. When using a
sequence comparison algorithm, test and reference sequences are
input into a computer, subsequence coordinates are designated, if
necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0114] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by visual
inspection (see generally Ausubel et al., supra).
[0115] Another example algorithm that is suitable for determining
percent sequence identity and sequence similarity is the BLAST
algorithm, which is described in Altschul et al., J. Mol. Biol.
215:403-410 (1990). Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information (at the web address www.ncbi.nlm.nih.gov). This
algorithm involves first identifying high scoring sequence pairs
(HSPs) by identifying short words of length W in the query
sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al., supra.). These initial neighborhood
word hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are then extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. For
identifying whether a nucleic acid or polypeptide is within the
scope of the invention, the default parameters of the BLAST
programs are suitable. The BLASTN program (for nucleotide
sequences) uses as defaults a word length (W) of 11, an expectation
(E) of 10, M=5, N=-4, and a comparison of both strands. For amino
acid sequences, the BLASTP program uses as defaults a word length
(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring
matrix. The TBLATN program (using protein sequence for nucleotide
sequence) uses as defaults a word length (W) of 3, an expectation
(E) of 10, and a BLOSUM 62 scoring matrix. (see Henikoff &
Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
[0116] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul,
Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.1, more preferably less than about 0.01, and
most preferably less than about 0.001.
[0117] Other considerations affecting the selection of
microorganisms for use in the invention include, in addition to
production of suitable lipids or hydrocarbons for production of
oils, fuels, and oleochemicals: (1) high lipid content as a
percentage of cell weight; (2) ease of growth; and (3) ease of
biomass processing. In particular embodiments, microorganism yields
cells that are at least 40%, at least 45%, at least 50%, at least
55%, at least 60%, at least 65%, or at least 70% or more lipid.
Preferred organisms grow heterotrophically (on sugars in the
absence of light).
[0118] B. Bioreactor
[0119] Microrganisms are cultured both for purposes of conducting
genetic manipulations and for production of hydrocarbons (e.g.,
lipids, fatty acids, aldehydes, alcohols, and alkanes). The former
type of culture is conducted on a small scale and initially, at
least, under conditions in which the starting microorganism can
grow. Culture for purposes of hydrocarbon production is usually
conducted on a large scale (e.g., 10,000 L, 40,000 L, 100,000 L or
larger bioreactors) in a bioreactor. Prototheca are typically
cultured in the methods of the invention in liquid media within a
bioreactor. Typically, the bioreactor does not allow light to
enter.
[0120] The bioreactor or fermentor is used to culture microalgal
cells through the various phases of their physiological cycle.
Bioreactors offer many advantages for use in heterotrophic growth
and propagation methods. To produce biomass for use in food,
microalgae are preferably fermented in large quantities in liquid,
such as in suspension cultures as an example. Bioreactors such as
steel fermentors can accommodate very large culture volumes (40,000
liter and greater capacity bioreactors are used in various
embodiments of the invention). Bioreactors also typically allow for
the control of culture conditions such as temperature, pH, oxygen
tension, and carbon dioxide levels. For example, bioreactors are
typically configurable, for example, using ports attached to
tubing, to allow gaseous components, like oxygen or nitrogen, to be
bubbled through a liquid culture. Other culture parameters, such as
the pH of the culture media, the identity and concentration of
trace elements, and other media constituents can also be more
readily manipulated using a bioreactor.
[0121] Bioreactors can be configured to flow culture media though
the bioreactor throughout the time period during which the
microalgae reproduce and increase in number. In some embodiments,
for example, media can be infused into the bioreactor after
inoculation but before the cells reach a desired density. In other
instances, a bioreactor is filled with culture media at the
beginning of a culture, and no more culture media is infused after
the culture is inoculated. In other words, the microalgal biomass
is cultured in an aqueous medium for a period of time during which
the microalgae reproduce and increase in number; however,
quantities of aqueous culture medium are not flowed through the
bioreactor throughout the time period. Thus in some embodiments,
aqueous culture medium is not flowed through the bioreactor after
inoculation.
[0122] Bioreactors equipped with devices such as spinning blades
and impellers, rocking mechanisms, stir bars, means for pressurized
gas infusion can be used to subject microalgal cultures to mixing.
Mixing may be continuous or intermittent. For example, in some
embodiments, a turbulent flow regime of gas entry and media entry
is not maintained for reproduction of microalgae until a desired
increase in number of said microalgae has been achieved.
[0123] Bioreactor ports can be used to introduce, or extract,
gases, solids, semisolids, and liquids, into the bioreactor chamber
containing the microalgae. While many bioreactors have more than
one port (for example, one for media entry, and another for
sampling), it is not necessary that only one substance enter or
leave a port. For example, a port can be used to flow culture media
into the bioreactor and later used for sampling, gas entry, gas
exit, or other purposes. Preferably, a sampling port can be used
repeatedly without altering compromising the axenic nature of the
culture. A sampling port can be configured with a valve or other
device that allows the flow of sample to be stopped and started or
to provide a means of continuous sampling. Bioreactors typically
have at least one port that allows inoculation of a culture, and
such a port can also be used for other purposes such as media or
gas entry.
[0124] Bioreactors ports allow the gas content of the culture of
microalgae to be manipulated. To illustrate, part of the volume of
a bioreactor can be gas rather than liquid, and the gas inlets of
the bioreactor to allow pumping of gases into the bioreactor. Gases
that can be beneficially pumped into a bioreactor include air,
air/CO.sub.2 mixtures, noble gases, such as argon, and other gases.
Bioreactors are typically equipped to enable the user to control
the rate of entry of a gas into the bioreactor. As noted above,
increasing gas flow into a bioreactor can be used to increase
mixing of the culture.
[0125] Increased gas flow affects the turbidity of the culture as
well. Turbulence can be achieved by placing a gas entry port below
the level of the aqueous culture media so that gas entering the
bioreactor bubbles to the surface of the culture. One or more gas
exit ports allow gas to escape, thereby preventing pressure buildup
in the bioreactor. Preferably a gas exit port leads to a "one-way"
valve that prevents contaminating microorganisms from entering the
bioreactor.
[0126] C. Media
[0127] Microalgal culture media typically contains components such
as a fixed nitrogen source, a fixed carbon source, trace elements,
optionally a buffer for pH maintenance, and phosphate (typically
provided as a phosphate salt). Other components can include salts
such as sodium chloride, particularly for seawater microalgae.
Nitrogen sources include organic and inorganic nitrogen sources,
including, for example, without limitation, molecular nitrogen,
nitrate, nitrate salts, ammonia (pure or in salt form, such as,
(NH.sub.4).sub.2SO.sub.4 and NH.sub.4OH), protein, soybean meal,
cornsteep liquor, and yeast extract. Examples of trace elements
include zinc, boron, cobalt, copper, manganese, and molybdenum in,
for example, the respective forms of ZnCl.sub.2, H.sub.3BO.sub.3,
CoCl.sub.2.6H.sub.2O, CuCl.sub.2.2H.sub.2O, MnCl.sub.2.4H.sub.2O
and (NH.sub.4).sub.6Mo.sub.7O.sub.24.4H.sub.2O.
[0128] Microorganisms useful in accordance with the methods of the
present invention are found in various locations and environments
throughout the world. As a consequence of their isolation from
other species and their resulting evolutionary divergence, the
particular growth medium for optimal growth and generation of lipid
and/or hydrocarbon constituents can be difficult to predict. In
some cases, certain strains of microorganisms may be unable to grow
on a particular growth medium because of the presence of some
inhibitory component or the absence of some essential nutritional
requirement required by the particular strain of microorganism.
[0129] Solid and liquid growth media are generally available from a
wide variety of sources, and instructions for the preparation of
particular media that is suitable for a wide variety of strains of
microorganisms can be found, for example, online at
http://www.utex.org/, a site maintained by the University of Texas
at Austin, 1 University Station A6700, Austin, Tex., 78712-0183,
for its culture collection of algae (UTEX). For example, various
fresh water and salt water media include those described in PCT
Pub. No. 2008/151149, incorporated herein by reference.
[0130] In a particular example, Proteose Medium is suitable for
axenic cultures, and a 1 L volume of the medium (pH .about.6.8) can
be prepared by addition of 1 g of proteose peptone to 1 liter of
Bristol Medium. Bristol medium comprises 2.94 mM NaNO.sub.3, 0.17
mM CaCl.sub.2.2H.sub.2O, 0.3 mM MgSO.sub.4.7H.sub.2O, 0.43 mM, 1.29
mM KH.sub.2PO.sub.4, and 1.43 mM NaCl in an aqueous solution. For
1.5% agar medium, 15 g of agar can be added to 1 L of the solution.
The solution is covered and autoclaved, and then stored at a
refrigerated temperature prior to use. Another example is the
Prototheca isolation medium (PIM), which comprises 10 g/L
postassium hydrogen phthalate (KHP), 0.9 g/L sodium hydroxide, 0.1
g/L magnesium sulfate, 0.2 g/L potassium hydrogen phosphate, 0.3
g/L ammonium chloride, 10 g/L glucose 0.001 g/L thiamine
hydrochloride, 20 g/L agar, 0.25 g/L 5-fluorocytosine, at a pH in
the range of 5.0 to 5.2 (see Pore, 1973, App. Microbiology, 26:
648-649). Other suitable media for use with the methods of the
invention can be readily identified by consulting the URL
identified above, or by consulting other organizations that
maintain cultures of microorganisms, such as SAG, CCAP, or CCALA.
SAG refers to the Culture Collection of Algae at the University of
Gottingen (Gottingen, Germany), CCAP refers to the culture
collection of algae and protozoa managed by the Scottish
Association for Marine Science (Scotland, United Kingdom), and
CCALA refers to the culture collection of algal laboratory at the
Institute of Botany (T{hacek over (r)}ebo{hacek over (n)}, Czech
Republic). Additionally, U.S. Pat. No. 5,900,370 describes media
formulations and conditions suitable for heterotrophic fermentation
of Prototheca species.
[0131] For oil production, selection of a fixed carbon source is
important, as the cost of the fixed carbon source must be
sufficiently low to make oil production economical. Thus, while
suitable carbon sources include, for example, acetate, floridoside,
fructose, galactose, glucuronic acid, glucose, glycerol, lactose,
mannose, N-acetylglucosamine, rhamnose, sucrose, and/or xylose,
selection of feedstocks containing those compounds is an important
aspect of the methods of the invention. Suitable feedstocks useful
in accordance with the methods of the invention include, for
example, black liquor, corn starch, depolymerized cellulosic
material, milk whey, molasses, potato, sorghum, sucrose, sugar
beet, sugar cane, rice, and wheat. Carbon sources can also be
provided as a mixture, such as a mixture of sucrose and
depolymerized sugar beet pulp. The one or more carbon source(s) can
be supplied at a concentration of at least about 50 .mu.M, at least
about 100 .mu.M, at least about 500 .mu.M, at least about 5 mM, at
least about 50 mM, and at least about 500 mM, of one or more
exogenously provided fixed carbon source(s). Carbon sources of
particular interest for purposes of the present invention include
cellulose (in a depolymerized form), glycerol, sucrose, and
sorghum, each of which is discussed in more detail below.
[0132] In accordance with the present invention, microorganisms can
be cultured using depolymerized cellulosic biomass as a feedstock.
Cellulosic biomass (e.g., stover, such as corn stover) is
inexpensive and readily available; however, attempts to use this
material as a feedstock for yeast have failed. In particular, such
feedstocks have been found to be inhibitory to yeast growth, and
yeast cannot use the 5-carbon sugars produced from cellulosic
materials (e.g., xylose from hemi-cellulose). By contrast,
microalgae can grow on processed cellulosic material. Cellulosic
materials generally include about 40-60% cellulose; about 20-40%
hemicellulose; and 10-30% lignin.
[0133] Suitable cellulosic materials include residues from
herbaceous and woody energy crops, as well as agricultural crops,
i.e., the plant parts, primarily stalks and leaves, not removed
from the fields with the primary food or fiber product. Examples
include agricultural wastes such as sugarcane bagasse, rice hulls,
corn fiber (including stalks, leaves, husks, and cobs), wheat
straw, rice straw, sugar beet pulp, citrus pulp, citrus peels;
forestry wastes such as hardwood and softwood thinnings, and
hardwood and softwood residues from timber operations; wood wastes
such as saw mill wastes (wood chips, sawdust) and pulp mill waste;
urban wastes such as paper fractions of municipal solid waste,
urban wood waste and urban green waste such as municipal grass
clippings; and wood construction waste. Additional cellulosics
include dedicated cellulosic crops such as switchgrass, hybrid
poplar wood, and miscanthus, fiber cane, and fiber sorghum.
Five-carbon sugars that are produced from such materials include
xylose.
[0134] In another embodiment of the methods of the invention, the
carbon source is glycerol, including acidulated and non-acidulated
glycerol byproduct from biodiesel transesterification. In one
embodiment, the carbon source includes glycerol and at least one
other carbone source. In some cases, all of the glycerol and the at
least one other fixed carbon source are provided to the
microorganism at the beginning of the fermentation. In some cases,
the glycerol and the at least one other fixed carbon source are
provided to the microorganism simultaneously at a predetermined
ratio. In some cases, the glycerol and the at least one other fixed
carbon source are fed to the microbes at a predetermined rate over
the course of fermentation.
[0135] Some microalgae undergo cell division faster in the presence
of glycerol than in the presence of glucose (see PCT Pub. No.
2008/151149). In these instances, two-stage growth processes in
which cells are first fed glycerol to rapidly increase cell
density, and are then fed glucose to accumulate lipids can improve
the efficiency with which lipids are produced. The use of the
glycerol byproduct of the transesterification process provides
significant economic advantages when put back into the production
process. Other feeding methods are provided as well, such as
mixtures of glycerol and glucose. Feeding such mixtures also
captures the same economic benefits. In addition, the invention
provides methods of feeding alternative sugars to microalgae such
as sucrose in various combinations with glycerol.
[0136] In another embodiment of the methods of the invention, the
carbon source is sucrose, including a complex feedstock containing
sucrose, such as thick cane juice from sugar cane processing. In
one embodiment, the culture medium further includes at least one
sucrose utilization enzyme. In some cases, the culture medium
includes a sucrose invertase.
[0137] In one embodiment, the sucrose invertase enzyme is a
secrectable sucrose invertase enzyme encoded by an exogenous
sucrose invertase gene expressed by the population of
microorganisms.
[0138] Complex feedstocks containing sucrose include waste molasses
from sugar cane processing; the use of this low-value waste product
of sugar cane processing can provide significant cost savings in
the production of hydrocarbons and other oils. Another complex
feedstock containing sucrose that is useful in the methods of the
invention is sorghum, including sorghum syrup and pure sorghum.
Sorghum syrup is produced from the juice of sweet sorghum cane. Its
sugar profile consists of mainly glucose (dextrose), fructose and
sucrose.
[0139] D. Oil Production
[0140] For the production of oil in accordance with the methods of
the invention, it is preferable to culture cells in the dark, as is
the case, for example, when using extremely large (40,000 liter and
higher) fermentors that do not allow light to strike the culture.
Microalgae, including Prototheca species are grown and propagated
for the production of oil in a medium containing a fixed carbon
source and in the absence of light; such growth is known as
heterotrophic growth.
[0141] As an example, an inoculum of lipid-producing microalgal
cells are introduced into the medium; there is a lag period (lag
phase) before the cells begin to propagate. Following the lag
period, the propagation rate increases steadily and enters the log,
or exponential, phase. The exponential phase is in turn followed by
a slowing of propagation due to decreases in nutrients such as
nitrogen, increases in toxic substances, and quorum sensing
mechanisms. After this slowing, propagation stops, and the cells
enter a stationary phase or steady growth state, depending on the
particular environment provided to the cells. For obtaining lipid
rich biomass, the culture is typically harvested well after then
end of the exponential phase, which may be terminated early by
allowing nitrogen or another key nutrient (other than carbon) to
become depleted, forcing the cells to convert the carbon sources,
present in excess, to lipid. Culture condition parameters can be
manipulated to optimize total oil production, the combination of
lipid species produced, and/or production of a specific oil.
[0142] As discussed above, a bioreactor or fermentor is used to
allow cells to undergo the various phases of their growth cycle. As
an example, an inoculum of lipid-producing cells can be introduced
into a medium followed by a lag period (lag phase) before the cells
begin growth. Following the lag period, the growth rate increases
steadily and enters the log, or exponential, phase. The exponential
phase is in turn followed by a slowing of growth due to decreases
in nutrients and/or increases in toxic substances. After this
slowing, growth stops, and the cells enter a stationary phase or
steady state, depending on the particular environment provided to
the cells. Lipid production by cells disclosed herein can occur
during the log phase or thereafter, including the stationary phase
wherein nutrients are supplied, or still available, to allow the
continuation of lipid production in the absence of cell
division.
[0143] Preferably, microorganisms grown using conditions described
herein and known in the art comprise at least about 20% by weight
of lipid, preferably at least about 40% by weight, more preferably
at least about 50% by weight, and most preferably at least about
60% by weight. Process conditions can be adjusted to increase the
yield of lipids suitable for a particular use and/or to reduce
production cost. For example, in certain embodiments, a microalgae
is cultured in the presence of a limiting concentration of one or
more nutrients, such as, for example, nitrogen, phosphorous, or
sulfur, while providing an excess of fixed carbon energy such as
glucose. Nitrogen limitation tends to increase microbial lipid
yield over microbial lipid yield in a culture in which nitrogen is
provided in excess. In particular embodiments, the increase in
lipid yield is at least about: 10%, 50%, 100%, 200%, or 500%. The
microbe can be cultured in the presence of a limiting amount of a
nutrient for a portion of the total culture period or for the
entire period. In particular embodiments, the nutrient
concentration is cycled between a limiting concentration and a
non-limiting concentration at least twice during the total culture
period. Lipid content of cells can be increased by continuing the
culture for increased periods of time while providing an excess of
carbon, but limiting or no nitrogen.
[0144] Microalgal biomass with a high percentage of oil/lipid
accumulation by dry weight has been generated using a variety of
different methods of culture known in the art. Microalgal biomass
with a higher percentage of accumulated oil/lipid is useful in
accordance with the present invention. Li et al. describe Chlorella
vulgaris cultures with up to 56.6% lipid by DCW in stationary
cultures grown under autotrophic conditions (i.e., photosynthetic
growth conditions) using high iron concentrations (Li et al.,
Bioresource Technology 99(11):4717-22 (2008)). Rodolfi et al.
describe Nanochloropsis sp and Chaetoceros calcitrans cultures with
60% lipid DCW and 39.8% lipid DCW, respectively, grown in a
photobioreactor under nitrogen starvation conditions (Rodolfi et
al., Biotechnology & Bioengineering 102(1):100-112 (2008)).
Solovchenko et al. describe Parietochloris incise cultures with
approximately 30% lipid accumulation (DCW) when grown
phototrophically and under low nitrogen conditions (Solovchenko et
al., Journal of Applied Phcology 20:245-251 (2008)). Chlorella
protothecoides can produce up to 55% lipid (DCW) grown under
certain heterotrophic conditions with nitrogen starvation (Miao and
Wu, Bioresource Technology 97:841-846 (2006)). Other Chlorella
species including Chlorella emersonii, Chlorella sorokiniana, and
Chlorella minutissima have been described to have accumulated up to
63% oil (DCW) when grown in stirred tank bioreactors under
low-nitrogen media conditions (Illman et al., Enzyme and Microbial
Technology 27:631-635 (2000)). Still higher percent lipid
accumulation by DCW has been reported, including 70% lipid (DCW)
accumulation in Dumaliella tertiolecta cultures grown in increased
NaCl conditions (Takagi et al., Journal of Bioscience and
Bioengineering 101(3): 223-226 (2006)) and 75% lipid accumulation
in Botryococcus braunii cultures (Banerjee et al., Critical Reviews
in Biotechnology 22(3): 245-279 (2002)). These and similar methods
can be used for photosynthetic and heterotrophic growth of
microalgae to produce oil.
[0145] In another embodiment, lipid yield is increased by culturing
a lipid-producing microbe (e.g., microalgae) in the presence of one
or more cofactor(s) for a lipid pathway enzyme (e.g., a fatty acid
synthetic enzyme). Generally, the concentration of the cofactor(s)
is sufficient to increase microbial lipid (e.g., fatty acid) yield
over microbial lipid yield in the absence of the cofactor(s). In a
particular embodiment, the cofactor(s) are provided to the culture
by including in the culture a microbe (e.g., microalgae) containing
an exogenous gene encoding the cofactor(s). Alternatively,
cofactor(s) may be provided to a culture by including a microbe
(e.g., microalgae) containing an exogenous gene that encodes a
protein that participates in the synthesis of the cofactor. In
certain embodiments, suitable cofactors include any vitamin
required by a lipid pathway enzyme, such as, for example: biotin,
pantothenate. Genes encoding cofactors suitable for use in the
invention or that participate in the synthesis of such cofactors
are well known and can be introduced into microbes (e.g.,
microalgae), using contructs and techniques such as those described
above.
[0146] The specific examples of bioreactors, culture conditions,
and heterotrophic growth and propagation methods described herein
can be combined in any suitable manner to improve efficiencies of
microbial growth and lipid and/or protein production.
[0147] Microalgal biomass with a high percentage of oil/lipid
accumulation by dry weight has been generated using different
methods of culture, which are known in the art (see PCT Pub. No.
2008/151149). Microalgal biomass generated by the culture methods
described herein and useful in accordance with the present
invention comprises at least 10% microalgal oil by dry weight. In
some embodiments, the microalgal biomass comprises at least 25%, at
least 50%, at least 55%, or at least 60% microalgal oil by dry
weight. In some embodiments, the microalgal biomass contains from
10-90% microalgal oil, from 25-75% microalgal oil, from 40-75%
microalgal oil, or from 50-70% microalgal oil by dry weight.
[0148] The microalgal oil of the biomass described herein, or
extracted from the biomass for use in the methods and compositions
of the present invention can comprise glycerolipids with one or
more distinct fatty acid ester side chains. Glycerolipids are
comprised of a glycerol molecule esterified to one, two or three
fatty acid molecules, which can be of varying lengths and have
varying degrees of saturation. The length and saturation
characteristics of the fatty acid molecules (and the microalgal
oils) can be manipulated to modify the properties or proportions of
the fatty acid molecules in the microalgal oils of the present
invention via culture conditions. Thus, specific blends of algal
oil can be prepared either within a single species of algae by
mixing together the biomass or algal oil from two or more species
of microalgae, or by blending algal oil of the invention with oils
from other sources such as soy, rapeseed, canola, palm, palm
kernel, coconut, corn, waste vegetable, Chinese tallow, olive,
sunflower, cottonseed, chicken fat, beef tallow, porcine tallow,
microalgae, macroalgae, microbes, Cuphea, flax, peanut, choice
white grease, lard, Camelina sativa, mustard seed, cashew nut,
oats, lupine, kenaf, calendula, help, coffee, linseed (flax),
hazelnut, euphorbia, pumpkin seed, coriander, camellia, sesame,
safflower, rice, tung tree, cocoa, copra, pium poppy, castor beans,
pecan, jojoba, macadamia, Brazil nuts, avocado, petroleum, or a
distillate fraction of any of the preceding oils.
[0149] The oil composition, i.e., the properties and proportions of
the fatty acid consitutents of the glycerolipids, can also be
manipulated by combining biomass or oil from at least two distinct
species of microalgae. In some embodiments, at least two of the
distinct species of microalgae have different glycerolipid
profiles. The distinct species of microalgae can be cultured
together or separately as described herein, preferably under
heterotrophic conditions, to generate the respective oils.
Different species of microalgae can contain different percentages
of distinct fatty acid constituents in the cell's
glycerolipids.
[0150] Generally, Prototheca strains have very little or no fatty
acids with the chain length C8-C14. For example, Prototheca
moriformis (UTEX 1435), Prototheca krugani (UTEX 329), Prototheca
stagnora (UTEX 1442) and Prototheca zopfii (UTEX 1438) contains no
(or undectable amounts) C8 fatty acids, between 0-0.01% C10 fatty
acids, between 0.03-2.1% C12 fatty acids and between 1.0-1.7% C14
fatty acids. Prototheca strains have a lipid profile of at least
20% C16 fatty acids, at least 50% C18:1 fatty acids and at least 8%
C18:2 fatty acids. As a non-limiting example, strains and species
of Prototheca have a lipid profile of: C14:0 fatty acid,
1.3.+-.0.6%; C16:0 fatty acid, 23.+-.4%; C16:1, 1.0.+-.0.5%; C18:0
fatty acid, 3.5.+-.1.5%; C18:1 fatty acid 62.+-.5%; C18:2 fatty
acid, 8.5.+-.1.0%; and each other fatty acid, .gtoreq.1.0%.
[0151] Microalgal oil can also include other constituents produced
by the microalgae, or incorporated into the microalgal oil from the
culture medium. These other constituents can be present in varying
amount depending on the culture conditions used to culture the
microalgae, the species of microalgae, the extraction method used
to recover microalgal oil from the biomass and other factors that
may affect microalgal oil composition. Non-limiting examples of
such constituents include carotenoids, present from 0.1-0.4
micrograms/ml, chlorophyll present from 0-0.02 milligrams/kilogram
of oil, gamma tocopherol present from 0.4-0.6 milligrams/100 grams
of oil, and total tocotrienols present from 0.2-0.5 milligrams/gram
of oil.
[0152] The other constituents can include, without limitation,
phospholipids, tocopherols, tocotrienols, carotenoids (e.g.,
alpha-carotene, beta-carotene, lycopene, etc.), xanthophylls (e.g.,
lutein, zeaxanthin, alpha-cryptoxanthin and beta-crytoxanthin), and
various organic or inorganic compounds.
[0153] In some cases, the oil extracted from Prototheca species
comprises no more than 0.02 mg/kg chlorophyll. In some cases, the
oil extracted from Prototheca species comprises no more than 0.4
mcg/ml total carotenoids. In some cases the Prototheca oil
comprises between 0.40-0.60 milligrams of gamma tocopherol per 100
grams of oil. In other cases, the Prototheca oil comprises between
0.2-0.5 milligrams of total tocotrienols per gram of oil.
IV. TRANSESTERIFICATION OF LIPIDS AND LIPID-BEARING BIOMASS
[0154] In situ transesterification of TAGs to fatty acid alkyl
esters in accordance with the methods of the present invention can
be performed on biomass generated from the microbial cultures
described above. In some embodiments, the biomass may comprise
biomass combined from two or more cultures of distinct strains or
species of microorganisms.
[0155] In some methods of the invention, the microbial biomass is
first harvested from the culture medium and dried, and then
subjected to an optional biomass disruption process prior to
transesterification. In other methods of the invention, the
microbial biomass is subjected to a biomass disruption process
prior to drying and transesterification. In some methods,
harvesting the biomass comprises separating the cellular components
of the biomass from the water and cell culture media by, for
example, passing the contents of the cell culture bioreactor
through a screen or similar filtering apparatus. In some
embodiments, harvesting the biomass comprises processing the
cellular components of the cell culture into a paste or low
moisture-content composition.
[0156] A. Drying Methods
[0157] Drying the biomass generated from the cultured
microorganisms described herein removes water that would otherwise
be available as a substrate during the transesterification
reaction, described in greater detail below, leading to the
formation of free fatty acids, rather than the desired fatty acid
alkyl esters. The extent to which biomass used in the in situ
transesterification methods of the present invention must be dried
depends on the alcohol:biomass ratio used in the
transesterification process, the cost of the alcohol, and the cost
or other volume constraints placed on the size of the reaction
vessel in which the transesterfication is to be performed. As will
be appreciated, these factors, balanced against the costs of drying
the biomass, determine an "acceptable dryness" for the biomass.
[0158] In some embodiments, the biomass can be dried using a drum
dryer in which the biomass is rotated in a drum and dried with the
application of air, which may be heated to expedite the drying
process. In other embodiments, an oven or spray dryer can be used
to facilitate drying of the biomass. Alternatively, the biomass may
be dried via a lyophilization process. The lyophilization process
may summarily be described as a "freeze-drying" process, in which
the biomass is frozen in a freeze-drying chamber. The application
of a vacuum to the freeze-drying chamber results in sublimation
(primary drying) and desorption (secondary drying) of the water
from the biomass, resulting in a product suitable for further
processing as described below. In still other embodiments a
combination of the foregoing may be utilized to appropriately dry
the biomass for further processing in accordance with the methods
described herein.
[0159] B. Biomass Disruption Methods
[0160] In some embodiments it may be desirable to disrupt the
biomass prior to in situ transesterification to make the
intracellular contents of the microorganisms more readily
accessible to the alcohol and catalyst transesterification
reagents. This can help to facilitate the conversion of TAGs to
fatty acid alkyl esters or other molecules in accordance with the
methods of the invention.
[0161] In some methods of the invention, disruption of the biomass
can be accomplished prior to subjecting the biomass to one or more
of the drying processes described above. In other methods,
disruption of the biomass can follow such a drying process. In some
methods, water is removed from the biomass prior to or after
disruption of the biomass, with or without subjecting the biomass
to a drying process. Following growth, the microorganisms are
optionally isolated by centrifuging the culture medium to generate
a concentrated microbial biomass. Disruption of the biomass can be
accomplished by lysing the microbial cells to produce a lysate.
Cell lysis can be achieved by any convenient means including
heat-induced lysis, addition of a base, addition of an acid, via
the use of enzymes such as proteases or polysaccharide degradation
enzymes such as amylases, via the use of ultrasound, mechanical
lysis, via the use of osmotic shock, infection with a lytic virus,
and/or expression of one or more lytic genes. Lysis is performed to
release intracellular molecules which have been produced by the
microorganism. Each of these methods for lysing a microorganism can
be used as a single method or in combination.
[0162] The extent of cell disruption can be observed by microscopic
analysis. Using one or more of the methods described herein,
typically more than 70% cell breakage is observed. Preferably, cell
breakage is more than 80%, more preferably more than 90% and most
preferably about 100%.
[0163] In particular embodiments, the microorganism is lysed after
growth, for example to increase the exposure of cellular lipid to a
catalyst for transesterification such as a lipase or a chemical
catalyst, expressed as described below. The timing of lipase
expression (e.g., via an inducible promoter), cell lysis, and the
adjustment of transesterification reaction conditions (e.g.,
removal of water, addition of alcohol, etc.) can be adjusted to
optimize the yield of fatty acid esters from lipase-mediated
transesterification.
[0164] In one embodiment of the present invention, the process of
lysing a microorganism comprises heating a cellular suspension
containing the microorganisms. In this embodiment, the culture
medium containing the microorganisms (or a suspension of
microorganisms isolated from the culture medium) is heated until
the microorganisms, i.e., the cell walls and membranes of
microorganisms, degrade or breakdown. Typically, temperatures
applied are at least 50.degree. C. Higher temperatures, such as, at
least 60.degree. C., at least 70.degree. C., at least 80.degree.
C., at least 90.degree. C., at least 100.degree. C., at least
110.degree. C., at least 120.degree. C., at least 130.degree. C. or
higher are used for more efficient cell lysis. Lysing cells by heat
treatment can be performed by boiling the microorganism.
Alternatively, heat treatment (without boiling) can be performed in
an autoclave. The heat treated lysate may be cooled for further
treatment. Cell disruption can also be performed by steam
treatment, i.e., through addition of pressurized steam. Steam
treatment of microalgae for cell disruption is described, for
example, in U.S. Pat. No. 6,750,048. In some embodiments steam
treatment may be achieved by sparging steam into the fermentor and
maintaining the broth at a desired temperature for less than about
90 minutes, preferably less than about 60 minutes, and more
preferably less than about 30 minutes.
[0165] In another embodiment of the present invention, the process
of lysing a microorganism comprises adding a base to a cellular
suspension containing the microorganism. The base should be strong
enough to hydrolyze at least a portion of the proteinaceous
compounds of the microorganisms used. Bases which are useful for
solubilizing proteins are known in the art of chemistry. Exemplary
bases which are useful in these methods include, but are not
limited to, hydroxides, carbonates and bicarbonates of lithium,
sodium, potassium, calcium, and mixtures thereof. A preferred base
is KOH. In another embodiment of the present invention, the process
of lysing a microorganism comprises adding an acid to a cellular
suspension containing the microorganism.
[0166] In another embodiment of the present invention, the process
of lysing a microorganism comprises lysing the microorganism with
an enzyme. Enzymes for lysing a microorganism include proteases and
polysaccharide-degrading enzymes such as hemicellulase, pectinase,
cellulase, and driselase. A polysaccharide-degrading enzyme,
optionally from Chlorella or a Chlorella virus, is preferred. A
preferred pair of enzymes for lysing oil bearing biomass are
alcalase and mannaway (Novozymes).
[0167] In another embodiment of the present invention, the process
of lysing a microorganism is performed using ultrasound, i.e.,
sonication. Cells can also by lysed with high frequency sound. The
sound can be produced electronically and transported through a
metallic tip to an appropriately concentrated cellular suspension.
This sonication (or ultrasonication) disrupts cellular integrity
based on the creation of cavities in the cell suspension.
[0168] In another embodiment of the present invention, the process
of lysing a microorganism is performed by mechanical means. Cells
can be lysed mechanically and optionally homogenized to facilitate
lipid transesterification. For example, a pressure disrupter can be
used to pump a cell containing slurry through a restricted orifice
valve. High pressure (up to 1500 bar) is applied, followed by an
instant expansion through an exiting nozzle. Cell disruption is
accomplished by three different mechanisms: impingement on the
valve, high liquid shear in the orifice, and sudden pressure drop
upon discharge, causing an explosion of the cell. The method
releases intracellular molecules. Alternatively, a ball mill can be
used. In a ball mill, cells are agitated in suspension with small
abrasive particles, such as beads. Cells break because of shear
forces, grinding between beads, and collisions with beads. The
beads disrupt the cells to release cellular contents. Cells can
also be disrupted by shear forces, such as with the use of blending
(e.g., with a high speed or Waring blender), the french press, or
even centrifugation in case of weak cell walls.
[0169] In another embodiment of the present invention, the process
of lysing a microorganism is performed by applying an osmotic
shock.
[0170] In another embodiment of the present invention, the process
of lysing a microorganism is performed by steam treatment, i.e.,
through addition of pressurized steam. Steam treatment of
microalgae for cell disruption is described, for example, in U.S.
Pat. No. 6,750,048.
[0171] In another embodiment of the present invention, the process
of lysing a microorganism comprises infection of the microorganism
with a lytic virus. A wide variety of viruses are known to lyse
microorganisms suitable for use in the methods of the present
invention, and the selection and use of a particular lytic virus
for a particular microorganism is within the level of skill in the
art. For example, paramecium bursaria chlorella virus (PBCV-1) is
the prototype of a group (family Phycodnaviridae, genus
Chlorovirus) of large, icosahedral, plaque-forming, double-stranded
DNA viruses that replicate in, and lyse, certain unicellular,
eukaryotic chlorella-like green algae. Accordingly, any susceptible
microalgae, such as C. protothecoides, can be lysed by infecting
the culture with a suitable chlorella virus. Methods of infecting
species of Chlorella with a chlorella virus are known. See for
example Adv. Virus Res. 2006; 66:293-336; Virology, 1999 Apr. 25;
257(1):15-23; Virology, 2004 Jan. 5; 318(1):214-23; Nucleic Acids
Symp. Ser. 2000; (44):161-2; J. Virol. 2006 March; 80(5):2437-44;
and Annu. Rev. Microbiol. 1999; 53:447-94.
[0172] In another embodiment of the present invention, the process
of lysing a microorganism comprises autolysis. In this embodiment,
a microorganism useful in the methods of the invention is
genetically engineered to produce a lytic gene that will lyse the
microorganism. This lytic gene can be expressed using an inducible
promoter, so that the cells can first be grown to a desirable
density in a culture medium and then harvested, followed by
induction of the promoter to express the lytic gene to lyse the
cells. In one embodiment, the lytic gene encodes a
polysaccharide-degrading enzyme. In certain other embodiments, the
lytic gene is a gene from a lytic virus. Thus, for example, a lytic
gene from a Chlorella virus can be expressed in a Chlorella such as
C. protothecoides.
[0173] Expression of lytic genes is preferably done using an
inducible promoter, such as a promoter active in microalgae that is
induced by a stimulus such as the presence of a small molecule,
light, heat, and other stimuli. Lytic genes from chlorella viruses
are known. For example, see Virology 260, 308-315 (1999); FEMS
Microbiology Letters 180 (1999) 45-53; Virology 263, 376-387
(1999); and Virology 230, 361-368 (1997).
[0174] In another embodiment, lysis can be performed using an
expeller press. In this process, biomass is forced through a
screw-type device at high pressure, lysing the cells and causing
the intracellular lipid to be released and separated from the
protein and fiber (and other components) in the cells.
[0175] In particular embodiments, the microoganisms are lysed after
growth, for example to increase the exposure of cellular lipid to a
catalyst for transesterification such as a lipase, discussed below,
or a chemical catalyst. The timing of lipase expression (e.g., via
an inducible promoter), cell lysis, and the modification of
transesterification reaction conditions (e.g., removal of water,
addition of alcohol, etc.) can be adjusted to optimize the yield of
fatty acid esters from lipase-mediated transesterification.
[0176] C. Transesterification
[0177] Lipids produced by microorganisms as described above are
subjected to a process of transesterification in accordance with
the methods of the invention to generate a lipophilic phase
containing fatty acid alkyl esters and a hydrophilic phase
comprising cell material and glycerol. In some methods of the
invention, the lipophilic phase is then separated from the
hydrophilic cell material.
[0178] 1. General Chemical Process
[0179] Animal and plant oils are typically made of triacylglycerols
(TAGs), which are esters of free fatty acids with the trihydric
alcohol, glycerol. In transesterification, the glycerol in a TAG is
replaced with a lower alkyl alcohol such as methanol, ethanol or
isopropanol. A typical reaction scheme is as follows:
##STR00001##
[0180] In this scheme, the alcohol is deprotonated with a base to
make it a stronger nucleophile. Commonly, ethanol or methanol is
used in vast excess (up to 50-fold). Normally, this reaction will
proceed either exceedingly slowly or not at all. Heat, as well as
an acid or base, can be used to help speed the reaction. The acid
or base is not consumed by the transesterification reaction; thus,
they are not reactants but catalysts. Almost all biodiesel has
traditionally been produced using the base-catalyzed technique, as
it requires only low temperatures and pressures and produces over
98% conversion yield (provided the starting oil is low in moisture
and free fatty acids).
[0181] A special case of transesterification is glycerolysis or the
use of glycerol(glycerin) to break chemical bonds. The glycerolysis
reaction is usually catalyzed by the addition of an acid or a base.
Glycerolysis can be performed on simple esters, fats, free fatty
acids or TAGs, wherein the methyl esters react with excess glycerol
to form mono- and/or diglycerides, producing methanol as a
by-product. Mono- and diglycerides are useful as emulsifiers and
are commonly added to food products.
[0182] 2. Using Recombinant Lipases for Transesterification
[0183] Transesterification has also been carried out experimentally
using an enzyme, such as a lipase, instead of a base.
Lipase-catalyzed transesterification can be carried out, for
example, at a temperature between the room temperature and
80.degree. C., and a molar ratio of the TAG to the lower alcohol of
greater than 1:1, preferably about 3:1. Lipases suitable for use in
transesterification in accordance with the methods of the present
invention include, but are not limited to, those listed in Table 1.
Other examples of lipases useful for transesterification are found
in, e.g. U.S. Pat. Nos. 4,798,793; 4,940,845 5,156,963; 5,342,768;
5,776,741 and WO89/01032, each of which is incorporated herein by
reference. Such lipases include, but are not limited to, lipases
produced by microorganisms of Rhizopus, Aspergillus, Candida,
Mucor, Pseudomonas, Rhizomucor, Candida, and Humicola and pancreas
lipase.
TABLE-US-00001 TABLE 1 Lipases for use in transesterification.
Aspergillus niger lipase ABG73614, Candida antarctica lipase B
(novozym-435) CAA83122, Candida cylindracea lipase AAR24090,
Candida lipolytica lipase (Lipase L; Amano Pharmaceutical Co.,
Ltd.), Candida rugosa lipase (e.g., Lipase-OF; Meito Sangyo Co.,
Ltd.), Mucor miehei lipase (Lipozyme IM 20), Pseudomonas
fluorescens lipase AAA25882, Rhizopus japonicas lipase (Lilipase
A-10FG) Q7M4U7_1, Rhizomucor miehei lipase B34959, Rhizopus oryzae
lipase (Lipase F) AAF32408, Serratia marcescens lipase (SM Enzyme)
ABI13521, Thermomyces lanuginosa lipase CAB58509, Lipase P (Nagase
ChemteX Corporation), and Lipase QLM (Meito Sangyo Co., Ltd.,
Nagoya, Japan)
[0184] One challenge to using a lipase for the production of fatty
acid esters suitable for biodiesel is that the price of lipase is
much higher than the price of sodium hydroxide (NaOH) used by the
strong base process. This challenge has been addressed by using an
immobilized lipase, which can be recycled. However, the activity of
the immobilized lipase must be maintained after being recycled for
a minimum number of cycles to allow a lipase-based process to
compete with the strong base process in terms of the production
cost. Immobilized lipases are subject to poisoning by the lower
alcohols typically used in transesterification. U.S. Pat. No.
6,398,707 (issued Jun. 4, 2002 to Wu et al.), incorporated herein
by reference, describes methods for enhancing the activity of
immobilized lipases and regenerating immobilized lipases having
reduced activity. Some suitable methods include immersing an
immobilized lipase in an alcohol having a carbon atom number not
less than 3 for a period of time, preferably from 0.5-48 hours, and
more preferably from 0.5-1.5 hours. Some suitable methods also
include washing a deactivated immobilized lipase with an alcohol
having a carbon atom number not less than 3 and then immersing the
deactivated immobilized lipase in a vegetable oil for 0.5-48
hours.
[0185] In particular embodiments, a recombinant lipase is expressed
in the same microorganisms that produce the lipid on which the
lipase acts. Suitable recombinant lipases include those listed
above in Table 1 and/or those described under the GenBank Accession
numbers listed above in Table 1, or a polypeptide that has at least
70% amino acid identity with one of the lipases listed above in
Table 1 and that exhibits lipase activity. In additional
embodiments, the enzymatic activity is present in a sequence that
has at least about 75%, at least about 80%, at least about 85%, at
least about 90%, at least about 95%, or at least about 99% identity
with one of the above described sequences, all of which are hereby
incorporated by reference. DNA encoding the lipase and selectable
marker is preferably codon-optimized cDNA. Methods of recoding
genes for expression in microalgae are described in U.S. Pat. No.
7,135,290.
[0186] An exemplary vector design for expression of a lipase gene
in a microorganism such as a microalgae contains a gene encoding a
lipase in operable linkage with a promoter active in microalgae.
Alternatively, if the vector does not contain a promoter in
operable linkage with the lipase gene, the lipase gene can be
transformed into the cells such that it becomes operably linked to
an endogenous promoter at the point of vector integration. The
promoterless method of transformation has been demonstrated in
microalgae (see, for example, Plant Journal 14:4, (1998), pp.
441-447). The vector can also contain a second gene that encodes a
protein that imparts resistance to an antibiotic or herbicide,
i.e., a selectable marker. Optionally, one or both gene(s) is/are
followed by a 3' untranslated sequence containing a polyadenylation
signal. Expression cassettes encoding the two genes can be
physically linked in the vector or on separate vectors.
Co-transformation of microalgae can also be used, in which distinct
vector molecules are simultaneously used to transform cells (see,
for example, Protist 2004 December; 155(4):381-93). The transformed
cells can be optionally selected based upon the ability to grow in
the presence of the antibiotic or other selectable marker under
conditions in which cells lacking the resistance cassette would not
grow.
[0187] DNA encoding the lipase and selectable marker can be
codon-optimized cDNA. Methods of recoding genes for expression in
microalgae are described in U.S. Pat. No. 7,135,290. Additional
information is available at the web address
www.kazusa.or.jp/codon.
[0188] Many promoters are active in microalgae, including promoters
that are endogenous to the algae being transformed, as well as
promoters that are not endogenous to the algae being transformed
(i.e., promoters from other algae, promoters from higher plants,
and promoters from plant viruses or algae viruses). Exogenous
and/or endogenous promoters that are active in microalgae, and
antibiotic resistance genes functional in microalgae are known in
the art. The promoter used to express an exogenous gene can be the
promoter naturally linked to that gene or can be a heterologous
gene. Some promoters are active in more than one species of
microalgae. Other promoters are species-specific. Preferred
promoters include promoters such as RBCS2 from Chlamydomonas
reinhardtii and viral promoters, such as cauliflower mosaic virus
(CMV) and chlorella virus, which have been shown to be active in
multiple species of microalgae (see, for example, Plant Cell Rep.
2005 March; 23(10-11):727-35; J Microbiol. 2005 August;
43(4):361-5; Mar Biotechnol (NY). 2002 January; 4(1):63-73).
[0189] Promoters, cDNAs, and 3'UTRs, as well as other elements of
the vectors, can be generated through cloning techniques using
fragments isolated from native sources (see, for example, Molecular
Cloning: A Laboratory Manual, Sambrook et al. (3d edition, 2001,
Cold Spring Harbor Press; and U.S. Pat. No. 4,683,202).
Alternatively, elements can be generated synthetically using known
methods (see, for example, Gene 1995 Oct. 16; 164(1):49-53).
[0190] Cells can be transformed by any suitable technique
including, e.g., biolistics, electroporation, glass bead
transformation and silicon carbide whisker transformation.
[0191] In particular embodiments, the lipase is expressed in an
inducible and/or targeted manner. The use of an inducible promoter
to express a lipase gene permits production of the lipase after
growth of the microorganism when conditions have been adjusted, if
necessary, to enhance transesterification, for example, after
disruption of the cells, reduction of the water content of the
reaction mixture, and/or addition sufficient alcohol to drive
conversion of TAGs to fatty acid esters. Inducible promoters useful
in the invention include those that mediate transcription of an
operably linked gene in response to a stimulus, such as an
exogenously provided small molecule, temperature (heat or cold),
light, etc. Suitable promoters can activate transcription of an
essentially silent gene or upregulate, preferably substantially,
transcription of an operably linked gene that is transcribed at a
low level. In the latter case, the level of transcription of the
lipase preferably does not significantly interfere with the growth
of the microorganism in which it is expressed.
[0192] It can be advantageous, in particular embodiments, to target
expression of the lipase to one or more cellular compartments,
where it is sequestered from the majority of cellular lipids until
initiation of the transesterification reaction.
V. PRODUCING FUELS AND OLEOCHEMICALS WITH MICROBIAL OILS
[0193] Microbial oil can be isolated from microbial biomass (as
described, for example, for various strains of Chlorella and
Prototheca in the examples below) and chemically treated to produce
a variety of useful fuels and other chemicals.
[0194] The common international standard for biodiesel is EN 14214.
ASTM D6751 is the most common biodiesel standard referenced in the
United States and Canada. Germany uses DIN EN 14214 and the UK
requires compliance with BS EN 14214. Basic industrial tests to
determine whether the products conform to these standards typically
include gas chromatography, HPLC, and others. Biodiesel meeting the
quality standards is very non-toxic, with a toxicity rating
(LD.sub.50) of greater than 50 mL/kg.
[0195] Although biodiesel that meets the ASTM standards has to be
non-toxic, there can be contaminants which tend to crystallize
and/or precipitate and fall out of solution as sediment. Sediment
formation is particularly a problem when biodiesel is used at lower
temperatures. The sediment or precipitates may cause problems such
as decreasing fuel flow, clogging fuel lines, clogging filters,
etc. Processes are well-known in the art that specifically deal
with the removal of these contaminants and sediments in biodiesel
in order to produce a higher quality product. Examples for such
processes include, but are not limited to, pretreatment of the oil
to remove contaiminants such as phospholipids and free fatty acids
(e.g., degumming, caustic refining and silica adsorbant filtration)
and cold filtration. Cold filtration is a process that was
developed specifically to remove any particulates and sediments
that are present in the biodiesel after production. This process
cools the biodiesel and filters out any sediments or precipitates
that might form when the fuel is used at a lower temperature. Such
a process is well known in the art and is described in US Patent
Application Publication No. 2007-0175091. Suitable methods may
include cooling the biodiesel to a temperature of less than about
38.degree. C. so that the impurities and contaminants precipitate
out as particulates in the biodiesel liquid. Diatomaceous earth or
other filtering material may then added to the cooled biodiesel to
form a slurry, which may then filtered through a pressure leaf or
other type of filter to remove the particulates. The filtered
biodiesel may then be run through a polish filter to remove any
remaining sediments and diatomaceous earth, so as to produce the
final biodiesel product.
[0196] Subsequent processes may also be used if the biodiesel will
be used in particularly cold temperatures. Such processes include
winterization and fractionation. Both processes are designed to
improve the cold flow and winter performance of the fuel by
lowering the cloud point (the temperature at which the biodiesel
starts to crystallize). There are several approaches to winterizing
biodiesel. One approach is to blend the biodiesel with petroleum
diesel. Another approach is to use additives that can lower the
cloud point of biodiesel. Another approach is to remove saturated
methyl esters indiscriminately by mixing in additives and allowing
for the crystallization of saturates and then filtering out the
crystals. Fractionation selectively separates methyl esters into
individual components or fractions, allowing for the removal or
inclusion of specific methyl esters. Fractionation methods include
urea fractionation, solvent fractionation and thermal
distillation.
[0197] Another valuable fuel provided by the methods of the present
invention is renewable diesel, which comprises alkanes, such as
C16:0 and C18:0 and thus, are distinguishable from biodiesel. High
quality renewable diesel conforms to the ASTM D975 standard. The
lipids produced by the methods of the present invention can serve
as feedstock to produce renewable diesel. Thus, in another aspect
of the present invention, a method for producing renewable diesel
is provided. Renewable diesel can be produced by at least three
processes: hydrothermal processing (hydrotreating);
hydroprocessing; and indirect liquefaction. These processes yield
non-ester distillates. During these processes, triacylglycerides
produced and isolated as described herein, are converted to
alkanes.
[0198] In one embodiment, the method for producing renewable diesel
comprises (a) cultivating a lipid-containing microorganism using
methods disclosed herein (b) lysing the microorganism to produce a
lysate, (c) isolating lipid from the lysed microorganism, and (d)
deoxygenating and hydrotreating the lipid to produce an alkane,
whereby renewable diesel is produced. Lipids suitable for
manufacturing renewable diesel can be obtained via extraction from
microbial biomass using an organic solvent such as hexane, or via
other methods, such as those described in U.S. Pat. No. 5,928,696.
Some suitable methods may include mechanical pressing and
centrifuging.
[0199] In some methods, the microbial lipid is first cracked in
conjunction with hydrotreating to reduce carbon chain length and
saturate double bonds, respectively. The material is then
isomerized, also in conjunction with hydrotreating. The naptha
fraction can then be removed through distillation, followed by
additional distillation to vaporize and distill components desired
in the diesel fuel to meet a D975 standard while leaving components
that are heavier than desired for meeting a D 975 standard.
Hydrotreating, hydrocracking, deoxygenation and isomerization
methods of chemically modifying oils, including triglyceride oils,
are well known in the art. See for example European patent
applications EP1741768 (A1); EP1741767 (A1); EP1682466 (A1);
EP1640437 (A1); EP1681337 (A1); EP1795576 (A1); and U.S. Pat. Nos.
7,238,277; 6,630,066; 6,596,155; 6,977,322; 7,041,866; 6,217,746;
5,885,440; 6,881,873.
[0200] In one embodiment of the method for producing renewable
diesel, treating the lipid to produce an alkane is performed by
hydrotreating of the lipid composition. In hydrothermal processing,
typically, biomass is reacted in water at an elevated temperature
and pressure to form oils and residual solids. Conversion
temperatures are typically 300.degree. to 660.degree. F., with
pressure sufficient to keep the water primarily as a liquid, 100 to
170 standard atmosphere (atm). Reaction times are on the order of
15 to 30 minutes. After the reaction is completed, the organics are
separated from the water. Thereby a distillate suitable for diesel
is produced.
[0201] A renewable diesel, also known as "green diesel," can also
be produced from fatty acids by traditional hydroprocessing
technology. The triglyceride-containing oils can be hydroprocessed
either as co-feed with petroleum or as a dedicated feed. The
product is a diesel fuel that conforms with the ASTM D975
specification. Thus, in another embodiment of the method for
producing renewable diesel, treating the lipid composition to
produce an alkane is performed by hydroprocessing of the lipid
composition.
[0202] In some methods of making renewable diesel, the first step
of treating a triglyceride is hydroprocessing to saturate double
bonds, followed by deoxygenation at elevated temperature in the
presence of hydrogen and a catalyst. In some methods, hydrogenation
and deoxygenation occur in the same reaction. In other methods
deoxygenation occurs before hydrogenation. Isomerization is then
optionally performed, also in the presence of hydrogen and a
catalyst. Naphtha components are preferably removed through
distillation. For examples, see U.S. Pat. Nos. 5,475,160
(hydrogenation of triglycerides); 5,091,116 (deoxygenation,
hydrogenation and gas removal); 6,391,815 (hydrogenation); and
5,888,947 (isomerization).
[0203] One suitable method for the hydrogenation of triglycerides
includes preparing an aqueous solution of copper, zinc, magnesium
and lanthanum salts and another solution of alkali metal or
preferably, ammonium carbonate. The two solutions may be heated to
a temperature of about 20.degree. C. to about 85.degree. C. and
metered together into a precipitation container at rates such that
the pH in the precipitation container is maintained between 5.5 and
7.5 in order to form a catalyst. Additional water may be used
either initially in the precipitation container or added
concurrently with the salt solution and precipitation solution. The
resulting precipitate may then be thoroughly washed, dried,
calcined at about 300.degree. C. and activated in hydrogen at
temperatures ranging from about 100.degree. C. to about 400.degree.
C. One or more triglycerides may then be contacted and reacted with
hydrogen in the presence of the above-described catalyst in a
reactor. The reactor may be a trickle bed reactor, fixed bed
gas-solid reactor, packed bubble column reactor, continuously
stirred tank reactor, a slurry phase reactor, or any other suitable
reactor type known in the art. The process may be carried out
either batchwise or in continuous fashion. Reaction temperatures
are typically in the range of from about 170.degree. C. to about
250.degree. C. while reaction pressures are typically in the range
of from about 300 psig to about 2000 psig. Moreover, the molar
ratio of hydrogen to triglyceride in the process of the present
invention is typically in the range of from about 20:1 to about
700:1. The process is typically carried out at a weight hourly
space velocity (WHSV) in the range of from about 0.1 hr.sup.-1 to
about 5 hr.sup.-1. One skilled in the art will recognize that the
time period required for reaction will vary according to the
temperature used, the molar ratio of hydrogen to triglyceride, and
the partial pressure of hydrogen. The products produced by the such
hydrogenation processes include fatty alcohols, glycerol, traces of
paraffins and unreacted triglycerides. These products are typically
separated by conventional means such as, for example, distillation,
extraction, filtration, crystallization, and the like.
[0204] Petroleum refiners use hydroprocessing to remove impurities
by treating feeds with hydrogen. Hydroprocessing conversion
temperatures are typically 300.degree. to 700.degree. F. Pressures
are typically 40 to 100 atm. The reaction times are typically on
the order of 10 to 60 minutes. Solid catalysts are employed to
increase certain reaction rates, improve selectivity for certain
products, and optimize hydrogen consumption.
[0205] Suitable methods for the deoxygenation of an oil includes
heating an oil to a temperature in the range of from about
350.degree. F. to about 550.degree. F. and continuously contacting
the heated oil with nitrogen under at least pressure ranging from
about atmospeheric to above for at least about 5 minutes.
[0206] Suitable methods for isomerization includes using alkali
isomerization and other oil isomerization known in the art.
[0207] Hydrotreating and hydroprocessing ultimately lead to a
reduction in the molecular weight of the feed. In the case of
triglyceride-containing oils, the triglyceride molecule is reduced
to four hydrocarbon molecules under hydroprocessing conditions: a
propane molecule and three heavier hydrocarbon molecules, typically
in the C8 to C18 range.
[0208] Thus, in one embodiment, the product of the one or more
chemical reaction(s) is a straight chain alkane mixture that
comprises ASTM D975 renewable diesel. Production of hydrocarbons by
microorganisms is reviewed by Metzger et al. Appl Microbiol
Biotechnol (2005) 66: 486-496 and A Look Back at the U.S.
Department of Enetgy's Aquatic Species Program: Biodiesel from
Algae, NREL/TP-580-24190, John Sheehan, Terri Dunahay, John
Benemann and Paul Roessler (1998).
[0209] A traditional ultra-low sulfur diesel can be produced from
any form of biomass by a two-step process. First, the biomass is
converted to a syngas, a gaseous mixture rich in hydrogen and
carbon monoxide. Then, the syngas is catalytically converted to
liquids. Typically, the production of liquids is accomplished using
Fischer-Tropsch (FT) synthesis. This technology applies to coal,
natural gas, and heavy oils. Thus, in yet another preferred
embodiment of the method for producing renewable diesel, treating
the lipid composition to produce an alkane is performed by indirect
liquefaction of the lipid composition.
[0210] The present invention also provides methods to produce jet
fuel. Jet fuel is clear to straw colored. The most common fuel is
an unleaded/paraffin oil-based fuel classified as Aeroplane A-1,
which is produced to an internationally standardized set of
specifications. Jet fuel is a mixture of a large number of
different hydrocarbons, possibly as many as a thousand or more. The
range of their sizes (molecular weights or carbon numbers) is
restricted by the requirements for the product, for example,
freezing point or smoke point. Kerosone-type Aeroplane fuel
(including Jet A and Jet A-1) has a carbon number distribution
between about 8 and 16 carbon numbers. Wide-cut or naphta-type
Aeroplane fuel (including Jet B) typically has a carbon number
distribution between about 5 and 15 carbons.
[0211] Both Aeroplanes (Jet A and Jet B) may contain a number of
additives. Useful additives include, but are not limited to,
antioxidants, antistatic agents, corrosion inhibitors, and fuel
system icing inhibitor (FSII) agents. Antioxidants prevent gumming
and usually, are based on alkylated phenols, for example, AO-30,
AO-31, or AO-37. Antistatic agents dissipate static electricity and
prevent sparking. Stadis 450 with dinonylnaphthylsulfonic acid
(DINNSA) as the active ingredient, is an example. Corrosion
inhibitors, e.g., DCI-4A is used for civilian and military fuels
and DCI-6A is used for military fuels. FSII agents, include, e.g.,
Di-EGME.
[0212] In one embodiment of the invention, a jet fuel is produced
by blending algal fuels with existing jet fuel. The lipids produced
by the methods of the present invention can serve as feedstock to
produce jet fuel. Thus, in another aspect of the present invention,
a method for producing jet fuel is provided. Herewith two methods
for producing jet fuel from the lipids produced by the methods of
the present invention are provided: fluid catalytic cracking (FCC);
and hydrodeoxygenation (HDO).
[0213] Fluid Catalytic Cracking (FCC) is one method which is used
to produce olefins, especially propylene from heavy crude
fractions. The lipids produced by the method of the present
invention can be converted to olefins. The process involves flowing
the lipids produced through an FCC zone and collecting a product
stream comprised of olefins, which is useful as a jet fuel. The
lipids produced are contacted with a cracking catalyst at cracking
conditions to provide a product stream comprising olefins and
hydrocarbons useful as jet fuel.
[0214] In one embodiment, the method for producing jet fuel
comprises (a) cultivating a lipid-containing microorganism using
methods disclosed herein, (b) lysing the lipid-containing
microorganism to produce a lysate, (c) isolating lipid from the
lysate, and (d) treating the lipid composition, whereby jet fuel is
produced. In one embodiment of the method for producing a jet fuel,
the lipid composition can be flowed through a fluid catalytic
cracking zone, which, in one embodiment, may comprise contacting
the lipid composition with a cracking catalyst at cracking
conditions to provide a product stream comprising C.sub.2-C.sub.5
olefins.
[0215] In certain embodiments of this method, it may be desirable
to remove any contaminants that may be present in the lipid
composition. Thus, prior to flowing the lipid composition through a
fluid catalytic cracking zone, the lipid composition is pretreated.
Pretreatment may involve contacting the lipid composition with an
ion-exchange resin. The ion exchange resin is an acidic ion
exchange resin, such as Amberlyst.TM.-15 and can be used as a bed
in a reactor through which the lipid composition is flowed, either
upflow or downflow. Other pretreatments may include mild acid
washes by contacting the lipid composition with an acid, such as
sulfuric, acetic, nitric, or hydrochloric acid. Contacting is done
with a dilute acid solution usually at ambient temperature and
atmospheric pressure.
[0216] The lipid composition, optionally pretreated, is flowed to
an FCC zone where the hydrocarbonaceous components are cracked to
olefins. Catalytic cracking is accomplished by contacting the lipid
composition in a reaction zone with a catalyst composed of finely
divided particulate material. The reaction is catalytic cracking,
as opposed to hydrocracking, and is carried out in the absence of
added hydrogen or the consumption of hydrogen. As the cracking
reaction proceeds, substantial amounts of coke are deposited on the
catalyst. The catalyst is regenerated at high temperatures by
burning coke from the catalyst in a regeneration zone.
Coke-containing catalyst, referred to herein as "coked catalyst",
is continually transported from the reaction zone to the
regeneration zone to be regenerated and replaced by essentially
coke-free regenerated catalyst from the regeneration zone.
Fluidization of the catalyst particles by various gaseous streams
allows the transport of catalyst between the reaction zone and
regeneration zone. Methods for cracking hydrocarbons, such as those
of the lipid composition described herein, in a fluidized stream of
catalyst, transporting catalyst between reaction and regeneration
zones, and combusting coke in the regenerator are well known by
those skilled in the art of FCC processes. Exemplary FCC
applications and catalysts useful for cracking the lipid
composition to produce C.sub.2-C.sub.5 olefins are described in
U.S. Pat. Nos. 6,538,169, 7,288,685, which are incorporated in
their entirety by reference.
[0217] Suitable FCC catalysts generally comprise at least two
components that may or may not be on the same matrix. In some
embodiments, both two components may be circulated throughout the
entire reaction vessel. The first component generally includes any
of the well-known catalysts that are used in the art of fluidized
catalytic cracking, such as an active amorphous clay-type catalyst
and/or a high activity, crystalline molecular sieve. Molecular
sieve catalysts may be preferred over amorphous catalysts because
of their much-improved selectivity to desired products. IN some
preferred embodiments, zeolites may be used as the molecular sieve
in the FCC processes. Preferably, the first catalyst component
comprises a large pore zeolite, such as an Y-type zeolite, an
active alumina material, a binder material, comprising either
silica or alumina and an inert filler such as kaolin.
[0218] In one embodiment, cracking the lipid composition of the
present invention, takes place in the riser section or,
alternatively, the lift section, of the FCC zone. The lipid
composition is introduced into the riser by a nozzle resulting in
the rapid vaporization of the lipid composition. Before contacting
the catalyst, the lipid composition will ordinarily have a
temperature of about 149.degree. C. to about 316.degree. C.
(300.degree. F. to 600.degree. F.). The catalyst is flowed from a
blending vessel to the riser where it contacts the lipid
composition for a time of abort 2 seconds or less.
[0219] The blended catalyst and reacted lipid composition vapors
are then discharged from the top of the riser through an outlet and
separated into a cracked product vapor stream including olefins and
a collection of catalyst particles covered with substantial
quantities of coke and generally referred to as "coked catalyst."
In an effort to minimize the contact time of the lipid composition
and the catalyst which may promote further conversion of desired
products to undesirable other products, any arrangement of
separators such as a swirl arm arrangement can be used to remove
coked catalyst from the product stream quickly. The separator, e.g.
swirl arm separator, is located in an upper portion of a chamber
with a stripping zone situated in the lower portion of the chamber.
Catalyst separated by the swirl arm arrangement drops down into the
stripping zone. The cracked product vapor stream comprising cracked
hydrocarbons including light olefins and some catalyst exit the
chamber via a conduit which is in communication with cyclones. The
cyclones remove remaining catalyst particles from the product vapor
stream to reduce particle concentrations to very low levels. The
product vapor stream then exits the top of the separating vessel.
Catalyst separated by the cyclones is returned to the separating
vessel and then to the stripping zone. The stripping zone removes
adsorbed hydrocarbons from the surface of the catalyst by
counter-current contact with steam.
[0220] Low hydrocarbon partial pressure operates to favor the
production of light olefins. Accordingly, the riser pressure is set
at about 172 to 241 kPa (25 to 35 psia) with a hydrocarbon partial
pressure of about 35 to 172 kPa (5 to 25 psia), with a preferred
hydrocarbon partial pressure of about 69 to 138 kPa (10 to 20
psia). This relatively low partial pressure for hydrocarbon is
achieved by using steam as a diluent to the extent that the diluent
is 10 to 55 wt-% of lipid composition and preferably about 15 wt-%
of lipid composition. Other diluents such as dry gas can be used to
reach equivalent hydrocarbon partial pressures.
[0221] The temperature of the cracked stream at the riser outlet
will be about 510.degree. C. to 621.degree. C. (950.degree. F. to
1150.degree. F.). However, riser outlet temperatures above
566.degree. C. (1050.degree. F.) make more dry gas and more
olefins. Whereas, riser outlet temperatures below 566.degree. C.
(1050.degree. F.) make less ethylene and propylene. Accordingly, it
is preferred to run the FCC process at a preferred temperature of
about 566.degree. C. to about 630.degree. C., preferred pressure of
about 138 kPa to about 240 kPa (20 to 35 psia). Another condition
for the process is the catalyst to lipid composition ratio which
can vary from about 5 to about 20 and preferably from about 10 to
about 15.
[0222] In one embodiment of the method for producing a jet fuel,
the lipid composition is introduced into the lift section of an FCC
reactor. The temperature in the lift section will be very hot and
range from about 700.degree. C. (1292.degree. F.) to about
760.degree. C. (1400.degree. F.) with a catalyst to lipid
composition ratio of about 100 to about 150. It is anticipated that
introducing the lipid composition into the lift section will
produce considerable amounts of propylene and ethylene.
[0223] In another embodiment of the method for producing a jet fuel
using the lipid composition or the lipids produced as described
herein, the structure of the lipid composition or the lipids is
broken by a process referred to as hydrodeoxygenation (HDO). HDO
means removal of oxygen by means of hydrogen, that is, oxygen is
removed while breaking the structure of the material. Olefinic
double bonds are hydrogenated and any sulphur and nitrogen
compounds are removed. Sulphur removal is called
hydrodesulphurization (HDS). Pretreatment and purity of the raw
materials (lipid composition or the lipids) contribute to the
service life of the catalyst.
[0224] Generally in the HDO/HDS step, hydrogen is mixed with the
feed stock (lipid composition or the lipids) and then the mixture
is passed through a catalyst bed as a co-current flow, either as a
single phase or a two phase feed stock. After the HDO/MDS step, the
product fraction is separated and passed to a separate isomerzation
reactor. An isomerization reactor for biological starting material
is described in the literature (FI 100 248) as a co-current
reactor.
[0225] The process for producing a fuel by hydrogenating a
hydrocarbon feed, e.g., the lipid composition or the lipids herein,
can also be performed by passing the lipid composition or the
lipids as a co-current flow with hydrogen gas through a first
hydrogenation zone, and thereafter the hydrocarbon effluent is
further hydrogenated in a second hydrogenation zone by passing
hydrogen gas to the second hydrogenation zone as a counter-current
flow relative to the hydrocarbon effluent. Exemplary HDO
applications and catalysts useful for cracking the lipid
composition to produce C.sub.2-C.sub.5 olefins are described in
U.S. Pat. No. 7,232,935, which is incorporated in its entirety by
reference.
[0226] Typically, in the hydrodeoxygenation step, the structure of
the biological component, such as the lipid composition or lipids
herein, is decomposed, oxygen, nitrogen, phosphorus and sulphur
compounds, and light hydrocarbons as gas are removed, and the
olefinic bonds are hydrogenated. In the second step of the process,
i.e. in the so-called isomerization step, isomerzation is carried
out for branching the hydrocarbon chain and improving the
performance of the paraffin at low temperatures.
[0227] In the first step, i.e. HDO step, of the cracking process,
hydrogen gas and the lipid composition or lipids herein which are
to be hydrogenated are passed to a HDO catalyst bed system either
as co-current or counter-current flows, said catalyst bed system
comprising one or more catalyst bed(s), preferably 1-3 catalyst
beds. The HDO step is typically operated in a co-current manner. In
case of a HDO catalyst bed system comprising two or more catalyst
beds, one or more of the beds may be operated using the
counter-current flow principle. In the HDO step, the pressure
varies between 20 and 150 bar, preferably between 50 and 100 bar,
and the temperature varies between 200 and 500.degree. C.,
preferably in the range of 300-400.degree. C. In the HDO step,
known hydrogenation catalysts containing metals from Group VII
and/or VIB of the Periodic System may be used. Preferably, the
hydrogenation catalysts are supported Pd, Pt, Ni, NiMo or a CoMo
catalysts, the support being alumina and/or silica. Typically,
NiMo/Al.sub.2O.sub.3 and CoMo/Al.sub.2O.sub.3 catalysts are
used.
[0228] Prior to the HDO step, the lipid composition or lipids
herein may optionally be treated by prehydrogenation under milder
conditions thus avoiding side reactions of the double bonds. Such
prehydrogenation is carried out in the presence of a
prehydrogenation catalyst at temperatures of 50 400.degree. C. and
at hydrogen pressures of 1 200 bar, preferably at a temperature
between 150 and 250.degree. C. and at a hydrogen pressure between
10 and 100 bar. The catalyst may contain metals from Group VIII
and/or VIB of the Periodic System. Preferably, the prehydrogenation
catalyst is a supported Pd, Pt, Ni, NiMo or a CoMo catalyst, the
support being alumina and/or silica.
[0229] A gaseous stream from the HDO step containing hydrogen is
cooled and then carbon monoxide, carbon dioxide, nitrogen,
phosphorus and sulphur compounds, gaseous light hydrocarbons and
other impurities are removed therefrom. After compressing, the
purified hydrogen or recycled hydrogen is returned back to the
first catalyst bed and/or between the catalyst beds to make up for
the withdrawn gas stream. Water is removed from the condensed
liquid. The liquid is passed to the first catalyst bed or between
the catalyst beds.
[0230] After the HDO step, the product is subjected to an
isomerization step. It is substantial for the process that the
impurities are removed as completely as possible before the
hydrocarbons are contacted with the isomerization catalyst. The
isomerization step comprises an optional stripping step, wherein
the reaction product from the HDO step may be purified by stripping
with water vapour or a suitable gas such as light hydrocarbon,
nitrogen or hydrogen. The optional stripping step is carried out in
counter-current manner in a unit upstream of the isomerization
catalyst, wherein the gas and liquid are contacted with each other,
or before the actual isomerization reactor in a separate stripping
unit utilizing counter-current principle.
[0231] After the stripping step the hydrogen gas and the
hydrogenated lipid composition or lipids herein, and optionally an
n-paraffin mixture, are passed to a reactive isomerization unit
comprising one or several catalyst bed(s). The catalyst beds of the
isomerization step may operate either in co-current or
counter-current manner.
[0232] It is important for the process that the counter-current
flow principle is applied in the isomerization step. In the
isomerization step this is done by carrying out either the optional
stripping step or the isomerization reaction step or both in
counter-current manner. In the isomerzation step, the pressure
varies in the range of 20 150 bar, preferably in the range of 20
100 bar, the temperature being between 200 and 500.degree. C.,
preferably between 300 and 400.degree. C. In the isomerization
step, isomerization catalysts known in the art may be used.
Suitable isomerization catalysts contain molecular sieve and/or a
metal from Group VII and/or a carrier. Preferably, the
isomerization catalyst contains SAPO-11 or SAPO41 or ZSM-22 or
ZSM-23 or ferrierite and Pt, Pd or Ni and Al.sub.2O.sub.3 or
SiO.sub.2. Typical isomerization catalysts are, for example,
Pt/SAPO-11/Al.sub.2O.sub.3, Pt/ZSM-22/Al.sub.2O.sub.3,
Pt/ZSM-23/Al.sub.2O.sub.3 and Pt/SAPO-11/SiO.sub.2. The
isomerization step and the HDO step may be carried out in the same
pressure vessel or in separate pressure vessels. Optional
prehydrogenation may be carried out in a separate pressure vessel
or in the same pressure vessel as the HDO and isomerization
steps.
[0233] Thus, in one embodiment, the product of the one or more
chemical reactions is a straight chain alkane mixture that
comprises ASTM D1655 jet fuel. In some embodiments, the composition
comforming to the specification of ASTM 1655 jet fuel has a sulfur
content that is less than 10 ppm. In other embodiments, the
composition conforming to the specification of ASTM 1655 jet fuel
has a T10 value of the distillation curve of less than 205.degree.
C. In another embodiment, the composition conforming to the
specification of ASTM 1655 jet fuel has a final boiling point (FBP)
of less than 300.degree. C. In another embodiment, the composition
conforming to the specification of ASTM 1655 jet fuel has a flash
point of at least 38.degree. C. In another embodiment, the
composition conforming to the specification of ASTM 1655 jet fuel
has a density between 775K/m.sup.3 and 840K/m.sup.3. In yet another
embodiment, the composition conforming to the specification of ASTM
1655 jet fuel has a freezing point that is below -47.degree. C. In
another embodiment, the composition conforming to the specification
of ASTM 1655 jet fuel has a net Heat of Combustion that is at least
42.8 MJ/K. In another embodiment, the composition conforming to the
specification of ASTM 1655 jet fuel has a hydrogen content that is
at least 13.4 mass %. In another embodiment, the composition
conforming to the specification of ASTM 1655 jet fuel has a thermal
stability, as tested by quantitative gravimetric JFTOT at
260.degree. C., that is below 3 mm of Hg. In another embodiment,
the composition conforming to the specification of ASTM 1655 jet
fuel has an existent gum that is below 7 mg/dl.
[0234] Thus, the present invention discloses a variety of methods
in which chemical modification of microalgal lipid is undertaken to
yield products useful in a variety of industrial and other
applications. Examples of processes for modifying oil produced by
the methods disclosed herein include, but are not limited to,
hydrolysis of the oil, hydroprocessing of the oil, and
esterification of the oil. The modification of the microalgal oil
produces basic oleochemicals that can be further modified to
selected derivative oleochemicals for a desired function. In a
manner similar to that described above with reference to the fuel
producing processes these chemical modifications can also be
performed on oils generated from the microbial cultures described
herein. Examples of basic oleochemicals include, but are not
limited to, soaps, fatty acids, fatty acid methyl esters, and
glycerol. Examples of derivative oleochemicals include, but are not
limited to, fatty nitriles, esters, dimer acids, quats,
surfactants, fatty alkanolamides, fatty alcohol sulfates, resins,
emulsifiers, fatty alcohols, olefins, and higher alkanes.
[0235] Hydrolysis of the fatty acid constituents from the
glycerolipids produced by the methods of the invention yields free
fatty acids that can be derivatized to produce other useful
chemicals. Hydrolysis occurs in the presence of water and a
catalyst which may be either an acid or a base. The liberated free
fatty acids can be derivatized to yield a variety of products, as
reported in the following: U.S. Pat. Nos. 5,304,664 (Highly
sulfated fatty acids); 7,262,158 (Cleansing compositions);
7,115,173 (Fabric softener compositions); 6,342,208 (Emulsions for
treating skin); 7,264,886 (Water repellant compositions); 6,924,333
(Paint additives); 6,596,768 (Lipid-enriched ruminant feedstock);
and 6,380,410 (Surfactants for detergents and cleaners).
[0236] With regard to hydrolysis, in one embodiment of the
invention, a triglyceride oil is optionally first hydrolyzed in a
liquid medium such as water or sodium hydroxide so as to obtain
glycerol and soaps. There are various suitable triglyceride
hydrolysis methods, including, but not limited to, saponification,
acid hydrolysis, alkaline hydrolysis, enzymatic hydrolysis
(referred herein as splitting), and hydrolysis using hot-compressed
water. One skilled in the art will recognize that a triglyceride
oil need not be hydrolyzed in order to produce an oleochemical;
rather, the oil may be converted directly to the desired
oleochemical by other known process. For example, the triglyceride
oil may be directly converted to a methyl ester fatty acid through
esterification.
[0237] In some embodiments, catalytic hydrolysis of the oil
produced by methods disclosed herein occurs by splitting the oil
into glycerol and fatty acids. As discussed above, the fatty acids
may then be further processed through several other modifications
to obtained derivative oleochemicals. For example, in one
embodiment the fatty acids may undergo an amination reaction to
produce fatty nitrogen compounds. In another embodiment, the fatty
acids may undergo ozonolysis to produce mono- and
dibasic-acids.
[0238] In other embodiments hydrolysis may occur via the, splitting
of oils produced herein to create oleochemicals. In some preferred
embodiments of the invention, a triglyceride oil may be split
before other processes is performed. One skilled in the art will
recognize that there are many suitable triglyceride splitting
methods, including, but not limited to, enzymatic splitting and
pressure splitting.
[0239] Generally, enzymatic oil splitting methods use enzymes,
lipases, as biocatalysts acting on a water/oil mixture. Enzymatic
splitting then splits the oil or fat, respectively, is into
glycerol and free fatty acids. The glycerol may then migrates into
the water phase whereas the organic phase enriches with free fatty
acids.
[0240] The enzymatic splitting reactions generally take place at
the phase boundary between organic and aqueous phase, where the
enzyme is present only at the phase boundary. Triglycerides that
meet the phase boundary then contribute to or participate in the
splitting reaction. As the reaction proceeds, the occupation
density or concentration of fatty acids still chemically bonded as
glycerides, in comparison to free fatty acids, decreases at the
phase boundary so that the reaction is slowed down. In certain
embodiments, enzymatic splitting may occur at room temperature. One
of ordinary skill in the art would know the suitable conditions for
splitting oil into the desired fatty acids.
[0241] By way of example, the reaction speed can be accelerated by
increasing the interface boundary surface. Once the reaction is
complete, free fatty acids are then separated from the organic
phase freed from enzyme, and the residue which still contains fatty
acids chemically bonded as glycerides is fed back or recycled and
mixed with fresh oil or fat to be subjected to splitting. In this
manner, recycled glycerides are then subjected to a further
enzymatic splitting process. In some embodiments, the free fatty
acids are extracted from an oil or fat partially split in such a
manner. In that way, if the chemically bound fatty acids
(triglycerides) are returned or fed back into the splitting
process, the enzyme consumption can be drastically reduced.
[0242] The splitting degree is determined as the ratio of the
measured acid value divided by the theoretically possible acid
value which can be computed for a given oil or fat. Preferably, the
acid value is measured by means of titration according to standard
common methods. Alternatively, the density of the aqueous glycerol
phase can be taken as a measure for the splitting degree.
[0243] In one embodiment, the splitting process as described herein
is also suitable for splitting the mono-, di- and triglyceride that
are contained in the so-called soap-stock from the alkali refining
processes of the produced oils. In this manner, the soap-stock can
be quantitatively converted without prior saponification of the
neutral oils into the fatty acids. For this purpose, the fatty
acids being chemically bonded in the soaps are released, preferably
before splitting, through an addition of acid. In certain
embodiments, a buffer solution is used in addition to water and
enzyme for the splitting process.
[0244] In one embodiment, oils produced in accordance with the
methods of the invention can also be subjected to saponification as
a method of hydrolysis. Animal and plant oils are typically made of
triacylglycerols (TAGs), which are esters of fatty acids with the
trihydric alcohol, glycerol. In an alkaline hydrolysis reaction,
the glycerol in a TAG is removed, leaving three carboxylic acid
anions that can associate with alkali metal cations such as sodium
or potassium to produce fatty acid salts. In this scheme, the
carboxylic acid constituents are cleaved from the glycerol moiety
and replaced with hydroxyl groups. The quantity of base (e.g., KOH)
that is used in the reaction is determined by the desired degree of
saponification. If the objective is, for example, to produce a soap
product that comprises some of the oils originally present in the
TAG composition, an amount of base insufficient to convert all of
the TAGs to fatty acid salts is introduced into the reaction
mixture. Normally, this reaction is performed in an aqueous
solution and proceeds slowly, but may be expedited by the addition
of heat. Precipitation of the fatty acid salts can be facilitated
by addition of salts, such as water-soluble alkali metal halides
(e.g., NaCl or KCl), to the reaction mixture. Preferably, the base
is an alkali metal hydroxide, such as NaOH or KOH. Alternatively,
other bases, such as alkanolamines, including for example
triethanolamine and aminomethylpropanol, can be used in the
reaction scheme. In some cases, these alternatives may be preferred
to produce a clear soap product.
[0245] In some methods, the first step of chemical modification may
be hydroprocessing to saturate double bonds, followed by
deoxygenation at elevated temperature in the presence of hydrogen
and a catalyst. In other methods, hydrogenation and deoxygenation
may occur in the same reaction. In still other methods
deoxygenation occurs before hydrogenation. Isomerization may then
be optionally performed, also in the presence of hydrogen and a
catalyst. Finally, gases and naphtha components can be removed if
desired. For example, see U.S. Pat. Nos. 5,475,160 (hydrogenation
of triglycerides); 5,091,116 (deoxygenation, hydrogenation and gas
removal); 6,391,815 (hydrogenation); and 5,888,947
(isomerization).
[0246] In some embodiments of the invention, the triglyceride oils
are partially or completely deoxygenated. The deoxygenation
reactions form desired products, including, but not limited to,
fatty acids, fatty alcohols, polyols, ketones, and aldehydes. In
general, without being limited by any particular theory, the
deoxygenation reactions involve a combination of various different
reaction pathways, including without limitation: hydrogenolysis,
hydrogenation, consecutive hydrogenation-hydrogenolysis,
consecutive hydrogenolysis-hydrogenation, and combined
hydrogenation-hydrogenolysis reactions, resulting in at least the
partial removal of oxygen from the fatty acid or fatty acid ester
to produce reaction products, such as fatty alcohols, that can be
easily converted to the desired chemicals by further processing.
For example, in one embodiment, a fatty alcohol may be converted to
olefins through FCC reaction or to higher alkanes through a
condensation reaction.
[0247] One such chemical modification is hydrogenation, which is
the addition of hydrogen to double bonds in the fatty acid
constituents of glycerolipids or of free fatty acids. The
hydrogenation process permits the transformation of liquid oils
into semi-solid or solid fats, which may be more suitable for
specific applications.
[0248] Hydrogenation of oil produced by the methods described
herein can be performed in conjunction with one or more of the
methods and/or materials provided herein, as reported in the
following: U.S. Pat. Nos. 7,288,278 (Food additives or
medicaments); 5,346,724 (Lubrication products); 5,475,160 (Fatty
alcohols); 5,091,116 (Edible oils); 6,808,737 (Structural fats for
margarine and spreads); 5,298,637 (Reduced-calorie fat
substitutes); 6,391,815 (Hydrogenation catalyst and sulfur
adsorbent); 5,233,099 and 5,233,100 (Fatty alcohols); 4,584,139
(Hydrogenation catalysts); 6,057,375 (Foam suppressing agents); and
7,118,773 (Edible emulsion spreads).
[0249] One skilled in the art will recognize that various processes
may be used to hydrogenate carbohydrates. One suitable method
includes contacting the carbohydrate with hydrogen or hydrogen
mixed with a suitable gas and a catalyst under conditions
sufficient in a hydrogenation reactor to form a hydrogenated
product. The hydrogenation catalyst generally can include Cu, Re,
Ni, Fe, Co, Ru, Pd, Rh, Pt, Os, Ir, and alloys or any combination
thereof, either alone or with promoters such as W, Mo, Au, Ag, Cr,
Zn, Mn, Sn, B, P, Bi, and alloys or any combination thereof. Other
effective hydrogenation catalyst materials include either supported
nickel or ruthenium modified with rhenium. In an embodiment, the
hydrogenation catalyst also includes any one of the supports,
depending on the desired functionality of the catalyst. The
hydrogenation catalysts may be prepared by methods known to those
of ordinary skill in the art.
[0250] In some embodiments the hydrogenation catalyst includes a
supported Group VIII metal catalyst and a metal sponge material
(e.g., a sponge nickel catalyst). Raney nickel provides an example
of an activated sponge nickel catalyst suitable for use in this
invention. In other embodiment, the hydrogenation reaction in the
invention is performed using a catalyst comprising a nickel-rhenium
catalyst or a tungsten-modified nickel catalyst. One example of a
suitable catalyst for the hydrogenation reaction of the invention
is a carbon-supported nickel-rhenium catalyst.
[0251] In an embodiment, a suitable Raney nickel catalyst may be
prepared by treating an alloy of approximately equal amounts by
weight of nickel and aluminum with an aqueous alkali solution,
e.g., containing about 25 weight % of sodium hydroxide. The
aluminum is selectively dissolved by the aqueous alkali solution
resulting in a sponge shaped material comprising mostly nickel with
minor amounts of aluminum. The initial alloy includes promoter
metals (i.e., molybdenum or chromium) in the amount such that about
1 to 2 weight % remains in the formed sponge nickel catalyst. In
another embodiment, the hydrogenation catalyst is prepared using a
solution of ruthenium(III) nitrosylnitrate, ruthenium (III)
chloride in water to impregnate a suitable support material. The
solution is then dried to form a solid having a water content of
less than about 1% by weight. The solid may then be reduced at
atmospheric pressure in a hydrogen stream at 300.degree. C.
(uncalcined) or 400.degree. C. (calcined) in a rotary ball furnace
for 4 hours. After cooling and rendering the catalyst inert with
nitrogen, 5% by volume of oxygen in nitrogen is passed over the
catalyst for 2 hours.
[0252] In certain embodiments, the catalyst described includes a
catalyst support. The catalyst support stabilizes and supports the
catalyst. The type of catalyst support used depends on the chosen
catalyst and the reaction conditions. Suitable supports for the
invention include, but are not limited to, carbon, silica,
silica-alumina, zirconia, titania, ceria, vanadia, nitride, boron
nitride, heteropolyacids, hydroxyapatite, zinc oxide, chromia,
zeolites, carbon nanotubes, carbon fullerene and any combination
thereof.
[0253] The catalysts used in this invention can be prepared using
conventional methods known to those in the art. Suitable methods
may include, but are not limited to, incipient wetting, evaporative
impregnation, chemical vapor deposition, wash-coating, magnetron
sputtering techniques, and the like.
[0254] The conditions for which to carry out the hydrogenation
reaction will vary based on the type of starting material and the
desired products. One of ordinary skill in the art, with the
benefit of this disclosure, will recognize the appropriate reaction
conditions. In general, the hydrogenation reaction is conducted at
temperatures of 80.degree. C. to 250.degree. C., and preferably at
90.degree. C. to 200.degree. C., and most preferably at 100.degree.
C. to 150.degree. C. In some embodiments, the hydrogenation
reaction is conducted at pressures from 500 KPa to 14000 KPa.
[0255] The hydrogen used in the hydrogenolysis reaction of the
current invention may include external hydrogen, recycled hydrogen,
in situ generated hydrogen, and any combination thereof. As used
herein, the term "external hydrogen" refers to hydrogen that does
not originate from the biomass reaction itself, but rather is added
to the system from another source.
[0256] In some embodiments of the invention, it is desirable to
convert the starting carbohydrate to a smaller molecule that will
be more readily converted to desired higher hydrocarbons. One
suitable method for this conversion is through a hydrogenolysis
reaction. Various processes are known for performing hydrogenolysis
of carbohydrates. One suitable method includes contacting a
carbohydrate with hydrogen or hydrogen mixed with a suitable gas
and a hydrogenolysis catalyst in a hydrogenolysis reactor under
conditions sufficient to form a reaction product comprising smaller
molecules or polyols. As used herein, the term "smaller molecules
or polyols" includes any molecule that has a smaller molecular
weight, which can include a smaller number of carbon atoms or
oxygen atoms than the starting carbohydrate. In an embodiment, the
reaction products include smaller molecules that include polyols
and alcohols. Someone of ordinary skill in the art would be able to
choose the appropriate method by which to carry out the
hydrogenolysis reaction.
[0257] In some embodiments, a 5 and/or 6 carbon sugar or sugar
alcohol may be converted to propylene glycol, ethylene glycol, and
glycerol using a hydrogenolysis catalyst. The hydrogenolysis
catalyst may include Cr, Mo, W, Re, Mn, Cu, Cd, Fe, Co, Ni, Pt, Pd,
Rh, Ru, Ir, Os, and alloys or any combination thereof, either alone
or with promoters such as Au, Ag, Cr, Zn, Mn, Sn, Bi, B, O, and
alloys or any combination thereof. The hydrogenolysis catalyst may
also include a carbonaceous pyropolymer catalyst containing
transition metals (e.g., chromium, molybdemum, tungsten, rhenium,
manganese, copper, cadmium) or Group VIII metals (e.g., iron,
cobalt, nickel, platinum, palladium, rhodium, ruthenium, iridium,
and osmium). In certain embodiments, the hydrogenolysis catalyst
may include any of the above metals combined with an alkaline earth
metal oxide or adhered to a catalytically active support. In
certain embodiments, the catalyst described in the hydrogenolysis
reaction may include a catalyst support as described above for the
hydrogenation reaction.
[0258] The conditions for which to carry out the hydrogenolysis
reaction will vary based on the type of starting material and the
desired products. One of ordinary skill in the art, with the
benefit of this disclosure, will recognize the appropriate
conditions to use to carry out the reaction. In general, they
hydrogenolysis reaction is conducted at temperatures of 110.degree.
C. to 300.degree. C., and preferably at 170.degree. C. to
220.degree. C., and most preferably at 200.degree. C. to
225.degree. C. In some embodiments, the hydrogenolysis reaction is
conducted under basic conditions, preferably at a pH of 8 to 13,
and even more preferably at a pH of 10 to 12. In some embodiments,
the hydrogenolysis reaction is conducted at pressures in a range
between 60 KPa and 16500 KPa, and preferably in a range between
1700 KPa and 14000 KPa, and even more preferably between 4800 KPa
and 11000 KPa.
[0259] The hydrogen used in the hydrogenolysis reaction of the
current invention can include external hydrogen, recycled hydrogen,
in situ generated hydrogen, and any combination thereof.
[0260] In some embodiments, the reaction products discussed above
may be converted into higher hydrocarbons through a condensation
reaction in a condensation reactor. In such embodiments,
condensation of the reaction products occurs in the presence of a
catalyst capable of forming higher hydrocarbons. While not
intending to be limited by theory, it is believed that the
production of higher hydrocarbons proceeds through a stepwise
addition reaction including the formation of carbon-carbon, or
carbon-oxygen bond. The resulting reaction products include any
number of compounds containing these moieties, as described in more
detail below.
[0261] In certain embodiments, suitable condensation catalysts
include an acid catalyst, a base catalyst, or an acid/base
catalyst. As used herein, the term "acid/base catalyst" refers to a
catalyst that has both an acid and a base functionality. In some
embodiments the condensation catalyst can include, without
limitation, zeolites, carbides, nitrides, zirconia, alumina,
silica, aluminosilicates, phosphates, titanium oxides, zinc oxides,
vanadium oxides, lanthanum oxides, yttrium oxides, scandium oxides,
magnesium oxides, cerium oxides, barium oxides, calcium oxides,
hydroxides, heteropolyacids, inorganic acids, acid modified resins,
base modified resins, and any combination thereof. In some
embodiments, the condensation catalyst can also include a modifier.
Suitable modifiers include La, Y, Sc, P, B, Bi, Li, Na, K, Rb, Cs,
Mg, Ca, Sr, Ba, and any combination thereof. In some embodiments,
the condensation catalyst can also include a metal. Suitable metals
include Cu, Ag, Au, Pt, Ni, Fe, Co, Ru, Zn, Cd, Ga, In, Rh, Pd, Ir,
Re, Mn, Cr, Mo, W, Sn, Os, alloys, and any combination thereof.
[0262] In certain embodiments, the catalyst described in the
condensation reaction may include a catalyst support as described
above for the hydrogenation reaction. In certain embodiments, the
condensation catalyst is self-supporting. As used herein, the term
"self-supporting" means that the catalyst does not need another
material to serve as support. In other embodiments, the
condensation catalyst in used in conjunction with a separate
support suitable for suspending the catalyst. In an embodiment, the
condensation catalyst support is silica.
[0263] The conditions under which the condensation reaction occurs
will vary based on the type of starting material and the desired
products. One of ordinary skill in the art, with the benefit of
this disclosure, will recognize the appropriate conditions to use
to carry out the reaction. In some embodiments, the condensation
reaction is carried out at a temperature at which the
thermodynamics for the proposed reaction are favorable. The
temperature for the condensation reaction will vary depending on
the specific starting polyol or alcohol. In some embodiments, the
temperature for the condensation reaction is in a range from
80.degree. C. to 500.degree. C., and preferably from 125.degree. C.
to 450.degree. C., and most preferably from 125.degree. C. to
250.degree. C. In some embodiments, the condensation reaction is
conducted at pressures in a range between 0 Kpa to 9000 KPa, and
preferably in a range between 0 KPa and 7000 KPa, and even more
preferably between 0 KPa and 5000 KPa.
[0264] The higher alkanes formed by the invention include, but are
not limited to, branched or straight chain alkanes that have from 4
to 30 carbon atoms, branched or straight chain alkenes that have
from 4 to 30 carbon atoms, cycloalkanes that have from 5 to 30
carbon atoms, cycloalkenes that have from 5 to 30 carbon atoms,
aryls, fused aryls, alcohols, and ketones. Suitable alkanes
include, but are not limited to, butane, pentane, pentene,
2-methylbutane, hexane, hexene, 2-methylpentane, 3-methylpentane,
2,2,-dimethylbutane, 2,3-dimethylbutane, heptane, heptene, octane,
octene, 2,2,4-trimethylpentane, 2,3-dimethyl hexane,
2,3,4-trimethylpentane, 2,3-dimethylpentane, nonane, nonene,
decane, decene, undecane, undecene, dodecane, dodecene, tridecane,
tridecene, tetradecane, tetradecene, pentadecane, pentadecene,
nonyldecane, nonyldecene, eicosane, eicosene, uneicosane,
uneicosene, doeicosane, doeicosene, trieicosane, trieicosene,
tetraeicosane, tetraeicosene, and isomers thereof. Some of these
products may be suitable for use as fuels.
[0265] In some embodiments, the cycloalkanes and the cycloalkenes
are unsubstituted. In other embodiments, the cycloalkanes and
cycloalkenes are mono-substituted. In still other embodiments, the
cycloalkanes and cycloalkenes are multi-substituted. In the
embodiments comprising the substituted cycloalkanes and
cycloalkenes, the substituted group includes, without limitation, a
branched or straight chain alkyl having 1 to 12 carbon atoms, a
branched or straight chain alkylene having 1 to 12 carbon atoms, a
phenyl, and any combination thereof. Suitable cycloalkanes and
cycloalkenes include, but are not limited to, cyclopentane,
cyclopentene, cyclohexane, cyclohexene, methyl-cyclopentane,
methyl-cyclopentene, ethyl-cyclopentane, ethyl-cyclopentene,
ethyl-cyclohexane, ethyl-cyclohexene, isomers and any combination
thereof.
[0266] In some embodiments, the aryls formed are unsubstituted. In
another embodiment, the aryls formed are mono-substituted. In the
embodiments comprising the substituted aryls, the substituted group
includes, without limitation, a branched or straight chain alkyl
having 1 to 12 carbon atoms, a branched or straight chain alkylene
having 1 to 12 carbon atoms, a phenyl, and any combination thereof.
Suitable aryls for the invention include, but are not limited to,
benzene, toluene, xylene, ethyl benzene, para xylene, meta xylene,
and any combination thereof.
[0267] The alcohols produced in the invention have from 4 to 30
carbon atoms. In some embodiments, the alcohols are cyclic. In
other embodiments, the alcohols are branched. In another
embodiment, the alcohols are straight chained. Suitable alcohols
for the invention include, but are not limited to, butanol,
pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol,
dodecanol, tridecanol, tetradecanol, pentadecanol, hexadecanol,
heptyldecanol, octyldecanol, nonyldecanol, eicosanol, uneicosanol,
doeicosanol, trieicosanol, tetraeicosanol, and isomers thereof.
[0268] The ketones produced in the invention have from 4 to 30
carbon atoms. In an embodiment, the ketones are cyclic. In another
embodiment, the ketones are branched. In another embodiment, the
ketones are straight chained. Suitable ketones for the invention
include, but are not limited to, butanone, pentanone, hexanone,
heptanone, octanone, nonanone, decanone, undecanone, dodecanone,
tridecanone, tetradecanone, pentadecanone, hexadecanone,
heptyldecanone, octyldecanone, nonyldecanone, eicosanone,
uneicosanone, doeicosanone, trieicosanone, tetraeicosanone, and
isomers thereof.
[0269] Another such chemical modification is interesterification.
Naturally produced glycerolipids do not have a uniform distribution
of fatty acid constituents. In the context of oils,
interesterification refers to the exchange of acyl radicals between
two esters of different glycerolipids. The interesterification
process provides a mechanism by which the fatty acid constituents
of a mixture of glycerolipids can be rearranged to modify the
distribution pattern. Interesterification is a well-known chemical
process, and generally comprises heating (to about 200.degree. C.)
a mixture of oils for a period (e.g, 30 minutes) in the presence of
a catalyst, such as an alkali metal or alkali metal alkylate (e.g.,
sodium methoxide). This process can be used to randomize the
distribution pattern of the fatty acid constituents of an oil
mixture, or can be directed to produce a desired distribution
pattern. This method of chemical modification of lipids can be
performed on materials provided herein, such as extracted microbial
oil or microbial biomass with a percentage of dry cell weight as
lipid at least 20%.
[0270] Directed interesterification, in which a specific
distribution pattern of fatty acids is sought, can be performed by
maintaining the oil mixture at a temperature below the melting
point of some TAGs which might occur. This results in selective
crystallization of these TAGs, which effectively removes them from
the reaction mixture as they-crystallize. The process can be
continued until most of the fatty acids in the oil have
precipitated, for example. A directed interesterification process
can be used, for example, to produce a product with a lower calorie
content via the substitution of longer-chain fatty acids with
shorter-chain counterparts. Directed interesterification can also
be used to produce a product with a mixture of fats that can
provide desired melting characteristics and structural features
sought in food additives or products (e.g., margarine) without
resorting to hydrogenation, which can produce unwanted trans
isomers.
[0271] Interesterification of oils produced by the methods
described herein can be performed in conjuction with one or more of
the methods and/or materials, or to produce products, as reported
in the following: U.S. Pat. Nos. 6,080,853 (Nondigestible fat
substitutes); 4,288,378 (Peanut butter stabilizer); 5,391,383
(Edible spray oil); 6,022,577 (Edible fats for food products);
5,434,278 (Edible fats for food products); 5,268,192 (Low calorie
nut products); 5,258,197 (Reduce calorie edible compositions);
4,335,156 (Edible fat product); 7,288,278 (Food additives or
medicaments); 7,115,760 (Fractionation process); 6,808,737
(Structural fats); 5,888,947 (Engine lubricants); 5,686,131 (Edible
oil mixtures); and 4,603,188 (Curable urethane compositions).
[0272] In one embodiment in accordance with the invention,
transesterification of the oil, as described above, is followed by
reaction of the transesterified product with polyol, as reported in
U.S. Pat. No. 6,465,642, to produce polyol fatty acid polyesters.
Such an esterification and separation process may comprise the
steps as follows: reacting a lower alkyl ester with polyol in the
presence of soap; removing residual soap from the product mixture;
water-washing and drying the product mixture to remove impurities;
bleaching the product mixture for refinement; separating at least a
portion of the unreacted lower alkyl ester from the polyol fatty
acid polyester in the product mixture; and recycling the separated
unreacted lower alkyl ester.
[0273] Transesterification can also be performed on microbial
biomass with short chain fatty acid esters, as reported in U.S.
Pat. No. 6,278,006. In general, transesterification may be
performed by adding a short chain fatty acid ester to an oil in the
presence of a suitable catalyst and heating the mixture. In some
embodiments, the oil comprises about 5% to about 90% of the
reaction mixture by weight. In some embodiments, the short chain
fatty acid esters can be about 10% to about 50% of the reaction
mixture by weight. Non-limiting examples of catalysts include base
catalysts, sodium methoxide, acid catalysts including inorganic
acids such as sulfuric acid and acidified clays, organic acids such
as methane sulfonic acid, benzenesulfonic acid, and toluenesulfonic
acid, and acidic resins such as Amberlyst 15. Metals such as sodium
and magnesium, and metal hydrides also are useful catalysts.
[0274] Another such chemical modification is hydroxylation, which
involves the addition of water to a double bond resulting in
saturation and the incorporation of a hydroxyl moiety. The
hydroxylation process provides a mechanism for converting one or
more fatty acid constituents of a glycerolipid to a hydroxy fatty
acid. Hydroxylation can be performed, for example, via the method
reported in U.S. Pat. No. 5,576,027. Hydroxylated fatty acids,
including castor oil and its derivatives, are useful as components
in several industrial applications, including food additives,
surfactants, pigment wetting agents, defoaming agents, water
proofing additives, plasticizing agents, cosmetic emulsifying
and/or deodorant agents, as well as in electronics,
pharmaceuticals, paints, inks, adhesives, and lubricants. One
example of how the hydroxylation of a glyceride may be performed is
as follows: fat may be heated, preferably to about 30-50.degree. C.
combined with heptane and maintained at temperature for thirty
minutes or more; acetic acid may then be added to the mixture
followed by an aqueous solution of sulfuric acid followed by an
aqueous hydrogen peroxide solution which is added in small
increments to the mixture over one hour; after the aqueous hydrogen
peroxide, the temperature may then be increased to at least about
60.degree. C. and stirred for at least six hours; after the
stirring, the mixture is allowed to settle and a lower aqueous
layer formed by the reaction may be removed while the upper heptane
layer formed by the reaction may be washed with hot water having a
temperature of about 60.degree. C.; the washed heptane layer may
then be neutralized with an aqueous potassium hydroxide solution to
a pH of about 5 to 7 and then removed by distillation under vacuum;
the reaction product may then be dried under vacuum at 100.degree.
C. and the dried product steam-deodorized under vacuum conditions
and filtered at about 50.degree. to 60.degree. C. using
diatomaceous earth.
[0275] Hydroxylation of microbial oils produced by the methods
described herein can be performed in conjuction with one or more of
the methods and/or materials, or to produce products, as reported
in the following: U.S. Pat. Nos. 6,590,113 (Oil-based coatings and
ink); 4,049,724 (Hydroxylation process); 6,113,971 (Olive oil
butter); 4,992,189 (Lubricants and lube additives); 5,576,027
(Hydroxylated milk); and 6,869,597 (Cosmetics).
[0276] Hydroxylated glycerolipids can be converted to estolides.
Estolides consist of a glycerolipid in which a hydroxylated fatty
acid constituent has been esterified to another fatty acid
molecule. Conversion of hydroxylated glycerolipids to estolides can
be carried out by warming a mixture of glycerolipids and fatty
acids and contacting the mixture with a mineral acid, as described
by Isbell et al., JAOCS 71(2):169-174 (1994). Estolides are useful
in a variety of applications, including without limitation those
reported in the following: U.S. Pat. Nos. 7,196,124 (Elastomeric
materials and floor coverings); 5,458,795 (Thickened oils for
high-temperature applications); 5,451,332 (Fluids for industrial
applications); 5,427,704 (Fuel additives); and 5,380,894
(Lubricants, greases, plasticizers, and printing inks).
[0277] Other chemical reactions that can be performed on microbial
oils include reacting triacylglycerols with a cyclopropanating
agent to enhance fluidity and/or oxidative stability, as reported
in U.S. Pat. No. 6,051,539; manufacturing of waxes from
triacylglycerols, as reported in U.S. Pat. No. 6,770,104; and
epoxidation of triacylglycerols, as reported in "The effect of
fatty acid composition on the acrylation kinetics of epoxidized
triacylglycerols", Journal of the American Oil Chemists' Society,
79:1, 59-63, (2001) and Free Radical Biology and Medicine, 37:1,
104-114 (2004).
[0278] The generation of oil-bearing microbial biomass and the
extraction or separation of the oil for fuel and chemical products
as described above results in the production of delipidated biomass
meal. Delipidated meal is a byproduct of preparing algal oil and is
useful as animal feed for farm animals, e.g., ruminants, poultry,
swine and aquaculture. The resulting meal, although of reduced oil
content, still contains high quality proteins, carbohydrates,
fiber, ash, residual oil and other nutrients appropriate for an
animal feed. Because the cells are predominantly lysed by the oil
separation process, the delipidated meal is easily digestible by
such animals. Delipidated meal can optionally be combined with
other ingredients, such as grain, in an animal feed. Because
delipidated meal has a powdery consistency, it can be pressed into
pellets using an extruder or expander or another type of machine,
which are commercially available.
VI. OTHER METHODS OF CHEMICAL MODIFICATION OF LIPID-CONTAINING
BIOMASS
[0279] The present invention provides methods of chemical
modification of lipid-containing biomass other than
transesterification that yield products useful in a variety of
industrial and other applications. For example, the hydrogenation,
interesterification, hydroxylation, and hydrolysis plus
derivatization reactions described above in connection with making
fuels from microbial oil extracted or otherwise separated from
microbial biomass, can be carried out directly on high oil
containing microbial biomass in accordance with the methods of the
invention. Thus, in a manner similar to that described above with
reference to transesterification, these chemical modifications can
also be performed on biomass generated from the microbial cultures
described herein.
[0280] In some embodiments, the biomass may comprise biomass
combined from two or more cultures of distinct strains or species
of microorganisms. In some embodiments, the distinct strains or
species have different glycerolipid profiles. In some methods of
the invention, the microbial biomass is first harvested from the
culture medium, and then subjected to a chemical reaction that
covalently modifies at least 1% of the lipid. In some embodiments,
at least 2%, at least 3%, at least 4%, at least 5%, at least 10%,
at least 20%, at least 30%, at least 40%, at least 50%, at least
60%, at least 70%, at least 80%, or at least 90% of the lipid is
modified by the chemical process.
[0281] A. Hydrogenation: Saturation of Double Bonds
[0282] Hydrogenation is the addition of hydrogen to double bonds in
the fatty acid constituents of glycerolipids or of free fatty
acids. The hydrogenation process permits the transformation of
liquid oils into semi-solid or solid fats, which may be more
suitable for specific applications. Hydrogenation is a well-known
chemical process, and generally comprises contacting an oil mixture
with a finely divided transition metal (e.g., nickel, palladium,
platinum, or rhodium) catalyst at an elevated temperature (e.g.,
140-225.degree. C.) in the presence of hydrogen.
[0283] Hydrogenation of biomass produced by the methods described
herein can be performed in conjunction with one or more of the
methods and/or materials provided herein, including microbial
biomass with a percentage of DCW as lipid at least 20%, or to
produce products, as reported in the following: U.S. Pat. Nos.
7,288,278 (food additives or medicaments); 5,346,724 (lubrication
products); 5,475,160 (fatty alcohols); 5,091,116 (edible oils);
6,808,737 (structural fats for margarine and spreads); 5,298,637
(reduced-calorie fat substitutes); 6,391,815 (hydrogenation
catalyst and sulfur adsorbent); 5,233,099 and 5,233,100 (fatty
alcohols); 4,584,139 (hydrogenation catalysts); 6,057,375 (foam
suppressing agents); and 7,118,773 (edible emulsion spreads), each
of which is incorporated herein by reference.
[0284] B. Interesterification: Interchanging Fatty Acid Components
of Glycerolipids
[0285] Naturally produced glycerolipids typically do not have a
uniform distribution of fatty acid constituents. In the context of
oils, interesterification refers to the exchange of acyl radicals
between two esters of different glycerolipids. The
interesterification process provides a mechanism by which the fatty
acid constituents of a mixture of glycerolipids can be rearranged
to modify the distribution pattern. Interesterification is a
well-known chemical process, and generally comprises heating (to
about 200.degree. C.) a mixture of oils for a period (e.g, 30
minutes) in the presence of a catalyst, such as an alkali metal or
alkali metal alkylate (e.g., sodium methoxide). This process can be
used to randomize the distribution pattern of the fatty acid
constituents of an oil mixture, or can be directed to produce a
desired distribution pattern. This method of chemical modification
of lipids can be performed on materials provided herein, such as
microbial biomass with a lipid percentage of DCW of at least 20%.
Directed interesterification, in which a specific distribution
pattern of fatty acids is sought, can be performed by maintaining
the oil mixture at a temperature below the melting point of some
TAGs that might be present. This results in selective
crystallization of these TAGs, which effectively removes them from
the reaction mixture as they crystallize. The process can be
continued until most of the fatty acids in the oil have
precipitated. A directed interesterification process can be used to
produce, for example, a product with a lower calorie content via
the substitution of longer-chain fatty acids with shorter-chain
counterparts. Directed interesterification can also be used to
produce a product with a mixture of fats that can provide desired
melting characteristics and structural features sought in food
additives or food products (e.g., margarine) without resorting to
hydrogenation, which can produce unwanted trans isomers.
[0286] Interesterification of biomass produced by the methods
described herein can be performed in conjuction with one or more of
the methods and/or materials, or to produce products, as reported
in the following: U.S. Pat. Nos. 6,080,853 (nondigestible fat
substitutes); 4,288,378 (peanut butter stabilizer); 5,391,383
(edible spray oil); 6,022,577 (edible fats for food products);
5,434,278 (edible fats for food products); 5,268,192 (low calorie
nut products); 5,258,197 (reduced calorie edible compositions);
4,335,156 (edible fat product); 7,288,278 (food additives or
medicaments); 7,115,760 (fractionation process); 6,808,737
(structural fats); 5,888,947 (engine lubricants); 5,686,131 (edible
oil mixtures); and 4,603,188 (curable urethane compositions), each
of which is incorporated herein by reference. repeat of 289
above
[0287] In one embodiment of the invention, transesterification of
the biomass, as described above, is followed by reaction of the
transesterified product with polyol, as reported in U.S. Pat. No.
6,465,642, incorporated herein by reference, to produce polyol
fatty acid polyesters. Transesterification can also be performed on
microbial biomass with short chain fatty acid esters, as reported
in U.S. Pat. No. 6,278,006, incorporated herein by reference.
[0288] C. Hydroxylation: Saturation Via the Addition of Water to
Double Bonds
[0289] Hydroxylation involves the addition of water to a double
bond resulting in saturation and the incorporation of a hydroxyl
moiety. The hydroxylation process provides a mechanism for
converting one or more fatty acid constituents of a glycerolipid to
a hydroxy fatty acid. Hydroxylation can be performed, for example,
via the method reported in U.S. Pat. No. 5,576,027, incorporated
herein by reference. Hydroxylated fatty acids, including castor oil
and its derivatives, are useful as components in several industrial
applications, including as food additives, surfactants, pigment
wetting agents, defoaming agents, water proofing additives,
plasticizing agents, cosmetic emulsifying and/or deodorant agents,
as well as in electronics, pharmaceuticals, paints, inks,
adhesives, and lubricants.
[0290] Hydroxylation of microbial biomass produced by the methods
described herein can be performed in conjuction with one or more of
the methods and/or materials, or to produce products, as reported
in the following: U.S. Pat. Nos. 6,590,113 (oil-based coatings and
ink); 4,049,724 (hydroxylation process); 6,113,971 (olive oil
butter); 4,992,189 (lubricants and lube additives); 5,576,027
(hydroxylated milk); and 6,869,597 (cosmetics), each of which is
incorporated herein by reference.
[0291] Hydroxylated glycerolipids can be converted to estolides.
Estolides consist of a glycerolipid in which a hydroxylated fatty
acid constituent has been esterified to another fatty acid
molecule. Conversion of hydroxylated glycerolipids to estolides can
be carried out by warming a mixture of glycerolipids and fatty
acids and contacting the mixture with a mineral acid, as described
by Isbell et al., JAOCS 71(2):169-174 (1994), incorporated herein
by reference. Estolides are useful in a variety of applications,
including without limitation those reported in the following: U.S.
Pat. Nos. 7,196,124 (elastomeric materials and floor coverings);
5,458,795 (thickened oils for high-temperature applications);
5,451,332 (fluids for industrial applications); 5,427,704 (fuel
additives); and 5,380,894 (lubricants, greases, plasticizers, and
printing inks), each of which is incorporated herein by
reference.
[0292] D. Hydrolysis plus Derivatization: Cleavage and Modification
of Free Fatty Acids
[0293] Hydrolysis of the fatty acid constituents from the
glycerolipids produced by the methods of the invention yields free
fatty acids that can be derivatized to produce other useful
chemical entities. Hydrolysis occurs in the presence of water and
an acid or base catalyst. The liberated free fatty acids can be
derivatized to yield a variety of products, as reported in the
following: U.S. Pat. Nos. 5,304,664 (highly sulfated fatty acids);
7,262,158 (cleansing compositions); 7,115,173 (fabric softener
compositions); 6,342,208 (emulsions for treating skin); 7,264,886
(water repellant compositions); 6,924,333 (paint additives);
6,596,768 (lipid-enriched ruminant feedstock); and 6,380,410
(surfactants for detergents and cleaners), each of which is
incorporated herein by reference.
[0294] E. Additional Chemical Reactions to Modify Lipid-Containing
Microbial Biomass
[0295] Other chemical reactions that can be performed on
lipid-containing microbial biomass include reacting
triacylglycerols with a cyclopropanating agent to enhance fluidity
and/or oxidative stability, as reported in U.S. Pat. No. 6,051,539;
manufacturing of waxes from triacylglycerols, as reported in U.S.
Pat. No. 6,770,104; and epoxidation of triacylglycerols, as
reported in "The effect of fatty acid composition on the acrylation
kinetics of epoxidized triacylglycerols", Journal of the American
Oil Chemists' Society, 79:1, 59-63, (2001) and Free Radical Biology
and Medicine, 37:1, 104-114 (2004), each of which is incorporated
herein by reference.
[0296] In some methods, the first step of modification is
hydroprocessing to saturate double bonds, followed by deoxygenation
at elevated temperature in the presence of hydrogen and a catalyst.
In some methods, hydrogenation and deoxygenation occur in the same
reaction. In other methods deoxygenation occurs before
hydrogenation. Isomerization is then optionally performed, also in
the presence of hydrogen and a catalyst. Finally, gases and naphtha
components can be removed if desired. For example, see U.S. Pat.
Nos. 5,475,160 (hydrogenation of triglycerides); 5,091,116
(deoxygenation, hydrogenation and gas removal); 6,391,815
(hydrogenation); and 5,888,947 (isomerization), each of which is
incorporated herein by reference.
[0297] F. Saponification of Oil-Bearing Microbial Biomass and
Extracted Oil
[0298] 1. Basic Chemistry of Saponification
[0299] Animal and plant oils are typically made of triacylglycerols
(TAGs), which are esters of fatty acids with the trihydric alcohol,
glycerol. In an alkaline hydrolysis reaction, the glycerol in a TAG
is removed, leaving three carboxylic acid anions that can associate
with alkali metal cations such as sodium or potassium to produce
fatty acid salts. A typical reaction scheme is as follows:
##STR00002##
[0300] In this scheme, the carboxylic acid constituents are cleaved
from the glycerol moiety and replaced with hydroxyl groups. The
quantity of base (e.g., KOH) that is used in the reaction is
determined by the desired degree of saponifiction. If the objective
is, for example, to produce a soap product that comprises some of
the oils originally present in the TAG composition, an amount of
base insufficient to convert all of the TAGs to fatty acid salts is
introduced into the reaction mixture. Normally, this reaction is
performed in an aqueous solution and proceeds slowly, but may be
expedited by the addition of heat. Precipitation of the fatty acid
salts can be facilitated by addition of salts, such as
water-soluble alkali metal halides (e.g., NaCl or KCl), to the
reaction mixture. Preferably, the base is an alkali metal
hydroxide, such as NaOH or KOH. Alternatively, other bases, such as
alkanolamines, including for example triethanolamine and
aminomethylpropanol, can be used in the reaction scheme. In some
embodiments, these alternatives may be preferred to produce a clear
soap product.
[0301] 2. Saponification of Oil Bearing Biomass
[0302] Saponification of oil bearing microbial biomass can be
performed in accordance with the methods of the invention on intact
biomass or biomass that has been disrupted prior to being subjected
to the alkaline hydrolysis reaction. In the former case, intact
microbial biomass generated via the culturing of microorganisms as
described herein can be directly contacted with a base to convert
ester-containing lipid components of the biomass to fatty acid
salts. In some embodiments, all or a portion of the water in which
the microbes have been cultured is removed and the biomass is
resuspended in an aqueous solution containing an amount of base
sufficient to saponify the desired portion of the glycerolipid and
fatty acid ester components of the biomass. In some embodiments,
less than 100% of the glycerolipids and fatty acid esters in the
biomass are converted to fatty acid salts.
[0303] In some methods of the invention, the biomass is disrupted
prior to being subjected to the alkaline hydrolysis reaction.
Disruption of the biomass can be accomplished via any one or more
of the methods described above for lysing cells, including
heat-induced lysis, mechanical lysis, or the like, to make the
intracellular contents of the microorganisms more readily
accessible to the base. This can help to facilitate the conversion
of TAGs or fatty acid esters to fatty acid salts. Although
acid-induced lysis can be used to disrupt the biomass prior to
saponification, other methods may be more desirable to reduce the
possibility that additional base will be consumed to neutralize any
remaining acid during the alkaline hydrolysis reaction, which may
impact the conversion efficiency to fatty acid salts. Because the
application of heat can expedite the alkaline hydrolysis reaction,
heat-induced lysis can be used prior to or during the
saponification reaction to produce the fatty acid salts.
[0304] In some embodiments of the invention, the biomass is not
subjected to any treatment, or any treatment other than disruption,
prior to being subjected to the alkaline hydrolysis reaction. In
some embodiments, prior enrichment of the biomass to increase the
ratio of lipid to non-lipid material in the biomass to more than
50% (or by more than 50%) by weight, is performed as described
herein. In other embodiments, the biomass is subjected to the
alkaline hydrolysis reaction without a step of prior enrichment. In
some embodiments, the biomass subjected to the alkaline hydrolysis
reaction contains components other than water in the same relative
proportions as the biomass at the point of harvesting. In those
embodiments in which substantially all of the water has been
removed, the biomass comprises a cellular emulsion or
substantially-dried emulsion concentrate.
[0305] Any of the microorganisms described herein can be used to
produce lipid-containing biomass for the production of saponified
oils. In some embodiments, the microorganisms can also impart other
characteristics to the saponified-oil compositions produced from
the methods described herein. For example, microalgae of different
species, as well as microalgae grown under different conditions,
vary in color, including green, yellow, orange, red, and the like.
Small quantities of the compounds that impart these colors to the
microalgae can be purposefully retained so that the resulting
saponified-oil compositions and thereby provide natural colorants.
In some embodiments, other constituents of the biomass, including
carotenoids and xanthophylls, can also be retained in small
quantities in the saponified-oil compositions.
[0306] The extent of saponification of the biomass can vary in the
methods of the invention. In some embodiments, it is desirable to
produce a saponified-oil composition that also includes
glycerolipid constituents of the biomass. The appropriate quantity
of base (e.g., NaOH) for use in the alkaline hydrolysis reaction
can be determined based on an analysis of the glycerolipid and
fatty acid ester content of the biomass. In some embodiments, it is
preferable to use an excess of base to saponify lipid-containing
biomass directly, because some of the base may be consumed by
reaction with other constituents of the biomass. In some
embodiments, the use of excess quantities of base to saponify the
ester-containing lipid constituents of the biomass results in a
saponified oil composition that is undesirably alkaline. In these
instances, the composition can be purified to reduce the alkalinity
of the composition by boiling the saponified oil composition in
water and re-precipitating the fatty acid salts via addition of
salts such as NaCl, KCl, or the like. The purified soap composition
can then be subjected to further processing, such as removing
excess water, introducing various additives into the soap
composition, molding the soap into bars or other shapes, and the
like.
[0307] In some embodiments, the fatty acid salts (also referred to
as saponified oils) generated from the methods described herein can
be combined with one or more additives selected from essential
oils, fragrance oils, flavor oils, botanicals, extracts, CO.sub.2
extracts, clays, colorants, titanium dioxide, micas, tinting herbs,
glitters, exfoliants, fruit seeds, fibers, grain powders, nut
meals, seed meals, oil beads, wax beads, herbs, hydrosols,
vitamins, milk powders, preservatives, antioxidants, tocopherols,
salts, sugars, vegetable oils, waxes, glycerin, sea vegetables,
nutritive oils, moisturizing oils, vegetable butters, propylene
glycol, parabens, honey, bees wax, aloe, polysorbate, cornstarch,
cocoa powder, coral powder, humectants, gums, emulsifying agents,
and thickeners, or any other additives described herein.
[0308] 3. Saponification of Extracted Oil
[0309] The degree of saponification of extracted lipid constituents
of the biomass can be more readily controlled because of a reduced
probability that the base will be consumed through interaction with
components other than glycerolipids or fatty acid esters present in
the extracted oil. Extraction of the lipid constituents can be
performed via conventional hexane-extraction procedures, or via an
oil-extraction or solventless-extraction procedure.
[0310] Conventional hexane-extraction (other suitable organic
solvents can also be used) generally comprises contacting the
biomass or lysate with hexane in an amount and for a period of time
sufficient to allow the lipid to form a solution with the hexane.
The mixture can then be filtered and the hexane removed by, for
example, rotoevaporation. Hexane extraction methods are well known
in the art.
[0311] Oil extraction includes the addition of an oil directly to a
lysate without prior separation of the lysate components. After
addition of the oil, the lysate separates either of its own accord
or as a result of centrifugation or the like into different layers.
The layers can include in order of decreasing density: a pellet of
heavy solids, an aqueous phase, an emulsion phase, and an oil
phase. The emulsion phase is an emulsion of lipids and aqueous
phase. Depending on the percentage of oil added with respect to the
lysate (w/w or v/v), the force of centrifugation, if any, volume of
aqueous media and other factors, either or both of the emulsion and
oil phases can be present. Incubation or treatment of the cell
lysate or the emulsion phase with the oil is performed for a time
sufficient to allow the lipid produced by the microorganism to
become solubilized in the oil to form a heterogeneous mixture.
[0312] In various embodiments, the oil used in the extraction
process is selected from the group consisting of oil from soy,
rapeseed, canola, palm, palm kernel, coconut, corn, waste vegetable
oil, Chinese tallow, olive, sunflower, cotton seed, chicken fat,
beef tallow, porcine tallow, microalgae, macroalgae, Cuphea, flax,
peanut, choice white grease (lard), Camelina sativa mustard
seedcashew nut, oats, lupine, kenaf, calendula, hemp, coffee,
linseed, hazelnut, euphorbia, pumpkin seed, coriander, camellia,
sesame, safflower, rice, tung oil tree, cocoa, copra, pium poppy,
castor beans, pecan, jojoba, jatropha, macadamia, Brazil nuts, and
avocado. The amount of oil added to the lysate is typically greater
than 5% (measured by v/v and/or w/w) of the lysate with which the
oil is being combined. Thus, a preferred v/v or w/w of the oil is
greater than 5%, or at least 6%, at least 7%, at least 10%, at
least 20%, at least 25%, at least 30%. at least 40%, at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, and at
least 95% of the cell lysate.
[0313] Lipids can also be extracted from a lysate via a solventless
extraction procedure without substantial or any use of organic
solvents or oils by cooling the lysate. In such methods, the lysate
is preferably produced by acid treatment in combination with above
room temperature. Sonication can also be used, particularly if the
temperature is between room temperature and 65.degree. C. Such a
lysate on centrifugation or settling can be separated into layers,
one of which is an aqueous:lipid layer (the "emulsion" layer).
Other layers can include a solid pellet, an aqueous layer, and a
lipid layer. Lipid can be extracted from the emulsion layer by
freeze thawing or otherwise cooling the emulsion. In such methods,
it is not necessary to add any organic solvent or oil. If any
solvent or oil is added, it can be below 5% v/v or w/w of the
lysate.
[0314] The separated or extracted lipids are then subjected to an
alkaline hydrolysis reaction as described above, in which the
amount of base added to the reaction mixture can be tailored to
saponify a desired amount of the glycerolipid and fatty acid ester
constituents of the lipid composition. A close approximation or
quantification of the amount of esterified lipid in the composition
can be used to tailor the amount of base needed to saponify a
specified portion of the oil, thereby providing an opportunity to
modulate the amount of unsaponified oil remaining in the resulting
composition. In some embodiments, at least 1%, at least 2%, at
least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at
least 8%, at least 9%, or at least 10% of the oil, by weight,
remains unsaponified in the resulting composition. In other
embodiments, it may be desirable to saponify all or substantially
all of the oil, such that the resulting composition contains no
more than 10%, no more than 9%, no more than 8%, no more than 7%,
no more than 6%, no more than 5%, no more than 4%, no more than 3%,
no more than 2%, no more than 1%, or no more than 0.5% unsaponified
oil by weight.
[0315] In various embodiments of the invention, the microbial
biomass or oil can contain lipids with varying carbon chain
lengths, and with varying levels of saturation. The characteristics
of the lipids can result from the natural glycerolipid profiles of
the one or more microorganism populations used to generate the
biomass or oil subjected to the saponification reaction, or can be
the result of lipid pathway engineering, as described herein, in
which transgenic strains of microorganisms that produce particular
lipids in greater proportions are produced.
[0316] The microbial biomass subjected to transesterification or
other chemical modification, as described herein, can optionally be
subjected to a process of prior enrichment that increases the ratio
of the lipids to the dry weight of the microbes. In some
embodiments, the ratio of lipids to non-lipid materials in the
biomass is increased by more than 10%, by more than 20%, by more
than 30%, by more than 40%, by more than 50%, by more than 60%, by
more than 70%, by more than 80%, by more than 90%, or by more than
100% by weight. In some methods of the invention, the biomass is
subjected to the chemical reaction without a step of prior
enrichment, or, in some embodiments, without a step of prior
enrichment that increases the ratio by more than 50%. Enrichment of
the ratio of lipids to non-lipid material can be accomplished by,
for example, the addition of lipids obtained from a source other
than the microbial biomass (e.g., from a second microbial biomass
culture, from a plant or seed-oil source, or the like). Whether or
not subjected to optional enrichment, the lipid component comprises
no more than 50%, no more than 60%, no more than 70%, no more than
80%, no more than 90%, or no more than 95% of the biomass subjected
to the chemical reaction, and preferably the lipid component
comprises no less than 15%, no less than 20%, no less than 30%, no
less than 35%, no less than 40%, or no less than 45% of the
biomass. In some embodiments, the harvested biomass comprises a
lipid content of at least 5%, at least 10%, at least 15%, at least
20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 45%, at least 50%, at least 55%, at least 60%, at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, or at least
90% by DCW.
[0317] In some embodiments, water is removed from the biomass prior
to subjecting the biomass to the saponification (or other chemical
modification) reaction. In some embodiments of the invention, the
microbial biomass is not subjected to any treatment, other than
removing water and/or lysis, prior to subjecting the biomass to the
saponification (or other chemical modification) reaction. In some
embodiments, the biomass subjected to the chemical reaction
contains components other than water in the same relative
proportions as the biomass at the point of harvesting from the
fermentation. In this context, "the same relative proportions"
means that the proportions of the components remain substantially
the same after having accounted for changes associated with the
cells' use or metabolic conversion of some components following
harvesting of the biomass, chemical conversion of some components
within the harvested biomass (without the application of exogenous
reagents or catalysts), the escape of gases from the harvested
biomass, and/or similar modifications of the relative proportions
that are not readily controllable. The phrase "the same relative
proportions" is also meant to account for some level of
experimental variability, e.g, .+-.5%.
[0318] In some methods of the invention, the covalently modified
lipid is separated from other components of the biomass following
chemical modification of the lipid. In some embodiments, separating
the lipid comprises a phase separation whereby the covalently
modified lipids form a lighter non-aqueous phase and components of
the biomass form one or more heavier phases. The lighter
non-aqueous phase can then be removed to isolate the covalently
modified lipid components. In some embodiments, separation of a
lipophilic phase containing the covalently modified lipids from
hydrophilic cell material of the biomass can be facilitated by
centrifugation or other techniques. The ratio of the covalently
modified lipid to the biomass from which it is separated can be
between 10% lipid to 90% biomass and 90% lipid to 10% biomass by
dry weight.
[0319] 4. Advantages of Biomass with Higher Saturated Oil Content
and Fewer Colored Impurities
[0320] Although biomass and/or extracted oil for use in the
saponification methods described herein can be derived from any one
of a number of microorganisms with varying glycerolipid profiles
and varying ratios of other constituents such as pigments, in a
preferred embodiment, the biomass and/or the extracted oil
comprises a relatively high ratio of saturated fatty acids within
the TAGs and a relatively low ratio of constituents that impart a
color to the oil (e.g., pigments). In one embodiment, the biomass
and/or extracted oil is derived from microalgae of the genus
Prototheca.
[0321] The saturation characteristics of the fatty acid
constituents of a saponified oil, as well as the presence of
colored constituents, impact the shelf-life of compositions
comprising the saponified oil, as well as their aesthetic
qualities. Saturated fatty acids are less prone to oxidation than
their unsaturated counterparts. Thus, use of saponified oils with a
relatively higher ratio of saturated:unsaturated fatty acid
constituents in the preparation of saponified oil products results
in a longer overall shelf-life and minimizes the development of
oxidation products, which often have an unpleasant odor. Similarly,
the relative absence of colored impurities, which, upon oxidation
tend to change the appearance of the saponified oil composition in
which they are incorporated, improves the aesthetic qualities of
the composition and consumer satisfaction with such products,
particularly over an extended shelf-life. Consumers of the
resulting soap tend to associate a particular color or lack of
color with a brand of soap and come to expect the same color of
product every time. The lack of color in the saponified oil allows
for more consistency in the resulting saponified oil.
[0322] Higher ratios of saturated fatty acids are particularly
advantageous in the preparation of saponified compositions,
discussed below, in which a portion of the glycerolipids within the
biomass (or the extracted oil) remains unsaponified. As discussed
previously, a percentage of the glycerolipids can remain unmodified
(unsaponified) by adjusting the quantity of base used in the
saponification reaction, thus producing a soap product that retains
some proportion of the originally present glycerolipids. The
presence of an excess of glycerolipids in a saponification reaction
is commonly referred to as "superfatting." The extra oils remaining
in the product following the saponification reaction impart
moisturizing properties to the composition, but like any oil, are
subject to oxidation, which can lead to the development of an
unpleasant-smelling composition. Use of a higher ratio of
saturated:unsaturated fatty acid constituents as the "superfatting"
components of the reaction mixture results in a product with a
relatively longer shelf-life and minimizes the production of
malodorous oxidative products.
[0323] In various embodiments, saturated fatty acid constituents
comprise from 1-100% of the ester-containing lipid components of
the microbial biomass or extracted oil subjected to an alkaline
hydrolysis reaction in accordance with the methods of the present
invention. In preferred embodiments, saturated fatty acid
constituents comprise at least 50%, at least 60%, at least 70%, at
least 80%, at least 90%, at least 95%, or at least 99% of the
ester-containing lipid components in the alkaline hydrolysis
reaction.
[0324] In some embodiments, color-generating impurities (e.g.,
carotenoids) are present in the microbial biomass or the extracted
oil at a concentration of no more than 500 ppm, no more than 250
ppm, no more than 100 ppm, no more than 75 ppm, or no more than 25
ppm. Color-generating impurities include carotenoids such as
lutein, beta carotene, zeaxanthin, astaxanthin and chlorophyll. In
other embodiments, the amount of chlorophyll that is present in the
microbial biomass or the extracted oil is less than 0.1 mg/kg, less
than 0.05 mg/kg, or less than 0.01 mg/kg.
[0325] In some embodiments, the microbial oil or soap, before or
after saponification, respectively, contains less than 60
micrograms, less than 59 micrograms, less than 58 micrograms, less
than 57 micrograms, less than 56 micrograms, less than 55
micrograms, less than 54 micrograms, less than 53 micrograms, less
than 52 micrograms, less than 51 micrograms, less than 50
micrograms, less than 49 micrograms, less than 48 micrograms, less
than 47 micrograms, less than 46 micrograms, less than 45
micrograms, less than 44 micrograms, less than 43 micrograms, less
than 42 micrograms, less than 41 micrograms, less than 40
micrograms, less than 39 micrograms, less than 38 micrograms, less
than 37 micrograms, less than 36 micrograms, less than 35
micrograms, less than 34 micrograms, less than 33 micrograms, less
than 32 micrograms, less than 31 micrograms, less than 30
micrograms, less than 29 micrograms, less than 28 micrograms, less
than 27 micrograms, less than 26 micrograms, less than 25
micrograms, less than 24 micrograms, less than 23 micrograms, less
than 22 micrograms, less than 21 micrograms, less than 20
micrograms, less than 19 micrograms, less than 18 micrograms, less
than 17 micrograms, less than 16 micrograms, less than 15
micrograms, less than 14 micrograms, less than 13 micrograms, less
than 12 micrograms, less than 11 micrograms, less than 10
micrograms, less than 9 micrograms, less than 8 micrograms, less
than 7 micrograms, less than 6 micrograms, less than 5 micrograms
or less than 4 micrograms carotenoids per gram of saponified
oil.
[0326] Microalgae of the genus Prototheca, including without
limitation, Prototheca wickerhamii, Prototheca stagnora, Prototheca
portoricensis, Prototheca moriformis, and Prototheca zopfii
naturally produce higher ratios of saturated lipid constituents, as
illustrated in Example 28. Moreover, oils extracted from microalgae
of the genus Prototheca generally include fewer color-generating
impurities, allowing for the production of colorless compositions
comprising the saponified oils. Thus, use of such microorganisms as
the source of biomass or oil for practicing saponification methods
in accordance with the present invention is preferred.
VII. EXAMPLES
Example 1
Methods for Culturing Prototheca
[0327] Unless otherwise noted, all strains described in this and
the following Examples were obtained from the University of Texas
Culture Collection of Algae (Austin, Tex.). In this Example,
Prototheca strains were cultivated to achieve a high percentage of
oil by dry cell weight. Cryopreserved cells were thawed at room
temperature and 500 ul of cells were added to 4.5 ml of medium (4.2
g/L K.sub.2HPO.sub.4, 3.1 g/L NaH.sub.2PO.sub.4, 0.24 g/L
MgSO.sub.4.7H.sub.2O, 0.25 g/L Citric Acid monohydrate, 0.025 g/L
CaCl.sub.2 2H.sub.2O, 2 g/L yeast extract) plus 2% glucose and
grown for 7 days at 28.degree. C. with agitation (200 rpm) in a
6-well plate. Dry cell weights were determined by centrifuging 1 ml
of culture at 14,000 rpm for 5 min in a pre-weighed Eppendorf tube.
The culture supernatant was discarded and the resulting cell pellet
washed with 1 ml of deionized water. The culture was again
centrifuged, the supernatant discarded, and the cell pellets placed
at -80.degree. C. until frozen. Samples were then lyophilized for
24 hrs and dry cell weights calculated. For determination of total
lipid in cultures, 3 ml of culture was removed and subjected to
analysis using an Ankom system (Ankom Inc., Macedon, N.Y.)
according to the manufacturer's protocol. Samples were subjected to
solvent extraction with an Amkom XT10 extractor according to the
manufacturer's protocol. Total lipid was determined as the
difference in mass between acid hydrolyzed dried samples and
solvent extracted, dried samples. Percent oil dry cell weight
measurements are shown in Table 2.
TABLE-US-00002 TABLE 2 Percent oil by dry cell weight Species
Strain % Oil Prototheca stagnora UTEX 327 13.14 Prototheca
moriformis UTEX 1441 18.02 Prototheca moriformis UTEX 1435
27.17
[0328] Prototheca strains were genotyped. Genomic DNA was isolated
from algal biomass as follows. Cells (approximately 200 mg) were
centrifuged from liquid cultures 5 minutes at 14,000.times.g. Cells
were then resuspended in sterile distilled water, centrifuged 5
minutes at 14,000.times.g and the supernatant discarded. A single
glass bead .about.2 mm in diameter was added to the biomass and
tubes were placed at -80.degree. C. for at least 15 minutes.
Samples were removed and 150 .mu.l of grinding buffer (1% Sarkosyl,
0.25 M Sucrose, 50 mM NaCl, 20 mM EDTA, 100 mM Tris-HCl, pH 8.0,
RNase A 0.5 ug/ul) was added. Pellets were resuspended by vortexing
briefly, followed by the addition of 40 ul of 5M NaCl. Samples were
vortexed briefly, followed by the addition of 66 .mu.l of 5% CTAB
(Cetyl trimethylammonium bromide) and a final brief vortex. Samples
were next incubated at 65.degree. C. for 10 minutes after which
they were centrifuged at 14,000.times.g for 10 minutes. The
supernatant was transferred to a fresh tube and extracted once with
300 .mu.l of Phenol:Chloroform:Isoamyl alcohol 12:12:1, followed by
centrifugation for 5 minutes at 14,000.times.g. The resulting
aqueous phase was transferred to a fresh tube containing 0.7 vol of
isopropanol (.about.190 pe, mixed by inversion and incubated at
room temperature for 30 minutes or overnight at 4.degree. C. DNA
was recovered via centrifugation at 14,000.times.g for 10 minutes.
The resulting pellet was then washed twice with 70% ethanol,
followed by a final wash with 100% ethanol. Pellets were air dried
for 20-30 minutes at room temperature followed by resuspension in
50 .mu.l of 10 mM TrisCl, 1 mM EDTA (pH 8.0).
[0329] Five .mu.l of total algal DNA, prepared as described above,
was diluted 1:50 in 10 mM Tris, pH 8.0. PCR reactions, final volume
20 .mu.l, were set up as follows. Ten .mu.l of 2.times.iProof HF
master mix (BIO-RAD) was added to 0.4 .mu.l primer SZ02613
(5'-TGTTGAAGAATGAGCCGGCGAC-3' (SEQ ID NO: 1) at 10 mM stock
concentration). This primer sequence runs from position 567-588 in
Gen Bank accession no. L43357 and is highly conserved in higher
plants and algal plastid genomes. This was followed by the addition
of 0.4 .mu.l primer SZ02615 (5'-CAGTGAGCTATTACGCACTC-3' (SEQ ID NO:
2) at 10 mM stock concentration). This primer sequence is
complementary to position 1112-1093 in Gen Bank accession no.
L43357 and is highly conserved in higher plants and algal plastid
genomes. Next, 5 .mu.l of diluted total DNA and 3.2 .mu.l dH.sub.2O
were added. PCR reactions were run as follows: 98.degree. C., 45'';
98.degree. C., 8''; 53.degree. C., 12''; 72.degree. C., 20'' for 35
cycles followed by 72.degree. C. for 1 min and holding at
25.degree. C. For purification of PCR products, 20 .mu.l of 10 mM
Tris, pH 8.0, was added to each reaction, followed by extraction
with 40 .mu.l of Phenol:Chloroform:isoamyl alcohol 12:12:1,
vortexing and centrifuging at 14,000.times.g for 5 minutes. PCR
reactions were applied to S-400 columns (GE Healthcare) and
centrifuged for 2 minutes at 3,000.times.g. Purified PCR products
were subsequently TOPO cloned into PCR8/GW/TOPO and positive clones
selected for on LB/Spec plates. Purified plasmid DNA was sequenced
in both directions using M13 forward and reverse primers. In total,
twelve Prototheca strains were selected to have their 23S rRNA DNA
sequenced and the sequences are listed in the Sequence Listing. A
summary of the strains and Sequence Listing Numbers is included
below. The sequences were analyzed for overall divergence from the
UTEX 1435 (SEQ ID NO: 7) sequence. Two pairs emerged (UTEX 329/UTEX
1533 and UTEX 329/UTEX 1440) as the most divergent. In both cases,
pairwise alignment resulted in 75.0% pairwise sequence identity.
The percent sequence identity to UTEX 1435 is also included
below.
TABLE-US-00003 % nt Species Strain identity SEQ ID NO. Prototheca
kruegani UTEX 329 75.2 SEQ ID NO: 3 Prototheca wickerhamii UTEX
1440 99 SEQ ID NO: 4 Prototheca stagnora UTEX 1442 75.7 SEQ ID NO:
5 Prototheca moriformis UTEX 288 75.4 SEQ ID NO: 6 Prototheca
moriformis UTEX 1439; 100 SEQ ID NO: 7 1441; 1435; 1437 Prototheca
wikerhamii UTEX 1533 99.8 SEQ ID NO: 8 Prototheca moriformis UTEX
1434 75.9 SEQ ID NO: 9 Prototheca zopfii UTEX 1438 75.7 SEQ ID NO:
10 Prototheca moriformis UTEX 1436 88.9 SEQ ID NO: 11
[0330] Lipid samples from a subset of the above-listed strains were
analyzed for lipid profile using FAME GC/FID detection methods.
Results are shown below in Table 3.
TABLE-US-00004 TABLE 3 Lipid profile for Prototheca species. Strain
C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 UTEX 0 12.01
0 0 50.33 17.14 0 0 0 327 UTEX 1.41 29.44 0.70 3.05 57.72 12.37
0.97 0.33 0 1441 UTEX 1.09 25.77 0 2.75 54.01 11.90 2.44 0 0
1435
Example 2
Analysis of Extracted Oil and Delipidated Biomass
[0331] A. Extraction of Oil from Microalgae Using an Expeller Press
and a Press Aid
[0332] Microalgal biomass containing 38% oil by DCW was dried using
a drum dryer resulting in resulting moisture content of 5-5.5%. The
biomass was fed into a French L250 press. 30.4 kg (67 lbs.) of
biomass was fed through the press and no oil was recovered. The
same dried microbial biomass combined with varying percentage of
switchgrass as a press aid was fed through the press. The
combination of dried microbial biomass and 20% w/w switchgrass
yielded the best overall percentage oil recovery. The pressed cakes
were then subjected to hexane extraction and the final yield for
the 20% switchgrass condition was 61.6% of the total available oil
(calculated by weight). Biomass with above 50% oil dry cell weight
did not require the use of a pressing aid such as switchgrass in
order to liberate oil.
[0333] Oil extracted from wildtype Prototheca moriformis UTEX 1435
(via solvent extraction or using an expeller press was analyzed for
carotenoids, chlorophyll, tocopherols, other sterols and
tocotrienols. The results are summarized below in Table 4.
TABLE-US-00005 TABLE 4 Carotenoid, chlorophyll, tocopherol/sterols
and tocotrienol analysis in oil extracted from Prototheca
moriformis (UTEX 1435). Pressed oil Solvent extracted (mcg/ml) oil
(mcg/ml) cis-Lutein 0.041 0.042 trans-Lutein 0.140 0.112
trans-Zeaxanthin 0.045 0.039 cis-Zeaxanthin 0.007 0.013
t-alpha-Crytoxanthin 0.007 0.010 t-beta-Crytoxanthin 0.009 0.010
t-alpha-Carotene 0.003 0.001 c-alpha-Carotene none detected none
detected t-beta-Carotene 0.010 0.009 9-cis-beta-Carotene 0.004
0.002 Lycopene none detected none detected Total Carotenoids 0.267
0.238 Chlorophyll <0.01 mg/kg <0.01 mg/kg Tocopherols and
Sterols Pressed oil Solvent extracted (mg/100 g) oil (mg/100 g)
gamma Tocopherol 0.49 0.49 Campesterol 6.09 6.05 Stigmasterol 47.6
47.8 Beta-sitosterol 11.6 11.5 Other sterols 445 446 Tocotrienols
Pressed oil Solvent extracted (mg/g) oil (mg/g) alpha Tocotrienol
0.26 0.26 beta Tocotrienol <0.01 <0.01 gamma Tocotrienol 0.10
0.10 detal Tocotrienol <0.01 <0.01 Total Tocotrienols 0.36
0.36
[0334] B. Monosaccharide Composition of Delipidated Prototheca
moriformis Biomass
[0335] Prototheca moriformis (UTEX 1435) was grown in conditions
and nutrient media (with 4% glucose) as described in Example 1
above. The microalgal biomass was then harvested and dried using a
drum dryer. The dried algal biomass was lysed and the oil extracted
using an expeller press as described above. The residual oil in the
pressed biomass was then solvent extracted using petroleum ether.
Residual petroleum ether was evaporated from the delipidated meal
using a Rotovapor (Buchi Labortechnik AG, Switzerland). Glycosyl
(monosaccharide) composition analysis was then performed on the
delipidated meal using combined gas chromatography/mass
spectrometry (GC/MS) of the per-O-trimethylsily (TMS) derivatives
of the monosaccharide methyl glycosides produced from the sample by
acidic methanolysis. A sample of delipidated meal was subjected to
methanolysis in 1M HCl in methanol at 80.degree. C. for
approximately 20 hours, followed by re-N-acetylation with pyridine
and acetic anhydride in methanol (for detection of amino sugars).
The samples were then per-O-trimethylsiylated by treatment with
Tri-Sil (Pierce) at 80.degree. C. for 30 minutes (see methods in
Merkle and Poppe (1994) Methods Enzymol. 230:1-15 and York et al.,
(1985) Methods Enzymol. 118:3-40). GC/MS analysis of the TMS methyl
glycosides was performed on an HP 6890 GC interfaced to a 5975b
MSD, using a All Tech EC-1 fused silica capillary column (30
m.times.0.25 mm ID). The monosaccharides were identified by their
retention times in comparison to standards, and the carbohydrate
character of these are authenticated by their mass spectra. 20
micrograms per sample of inositol was added to the sample before
derivatization as an internal standard. The monosaccharide profile
of the delipidated Prototheca moriformis (UTEX 1435) biomass is
summarized in Table 5 below. The total percent carbohydrate from
the sample was calculated to be 28.7%.
TABLE-US-00006 TABLE 5 Monosaccharide (glycosyl) composition
analysis of Prototheca moriformis (UTEX 1435) delipidated biomass.
Mole % (of total Mass (.mu.g) carbohydrate) Arabinose 0.6 1.2
Xylose n.d. n.d. Galacturonic acid (GalUA) n.d. n.d. Mannose 6.9
11.9 Galactose 14.5 25.2 Glucose 35.5 61.7 N Acetyl Galactosamine
(GalNAc) n.d. n.d. N Acetyl Glucosamine (GlcNAc) n.d. n.d. Heptose
n.d. n.d. 3 Deoxy-2-manno-2 Octulsonic n.d. n.d. acid (KDO) Sum 57
100 n.d. = none detected
[0336] The carbohydrate content and monosaccharide composition of
the delipidated meal makes it suitable for use as an animal feed or
as part of an animal feed formulation. Thus, in one aspect, the
present invention provides delipidated meal having the product
content set forth in the table above.
Example 3
Fuel Production
[0337] A. Production of Biodiesel from Prototheca Oil
[0338] Degummed oil from Prototheca moriformis UTEX 1435, produced
according to the methods described above, was subjected to
transesterification to produce fatty acid methyl esters. Results
are shown below:
The lipid profile of the oil was:
C10:0 0.02
C12:0 0.06
C14:0 1.81
C14.1 0.07
C16:0 24.53
C16:1 1.22
C18:0 2.34
C18:1 59.21
C18:2 8.91
C18:3 0.28
C20:0 0.23
C20:1 0.10
C20:1 0.08
C21:0 0.02
C22:0 0.06
C24:0 0.10
TABLE-US-00007 [0339] TABLE 6 Biodiesel profile from Prototheca
moriformis triglyceride oil. Method Test Result Units ASTM Cold
Soak Filterability of Filtration Time 120 sec D6751 A1 Biodiesel
Blend Fuels Volume Filtered 300 ml ASTM D93 Pensky-Martens Closed
Cup Procedure Used A Flash Point Corrected Flash 165.0 .degree. C.
Point ASTM Water and Sediment in Middle Sediment and Water 0.000
Vol % D2709 Distillate Fuels (Centrifuge Method) EN 14538
Determination of Ca and Mg Sum of (Ca and <1 mg/kg Content by
ICP OES Mg) EN 14538 Determination of Ca and Mg Sum of (Na and K)
<1 mg/kg Content by ICP OES ASTM D445 Kinematic/Dynamic
Kinematic Viscosity 4.873 mm.sup.2/s Viscosity @ 104.degree.
F./40.degree. C. ASTM D874 Sulfated Ash from Lubricating Sulfated
Ash <0.005 Wt % Oils and Additives ASTM Determination of Total
Sulfur Sulfur, mg/kg 1.7 mg/kg D5453 in Light Hydrocarbons, Spark
Ignition Engine Fuel, Diesel Engine Fuel, and Engine Oil by
Ultraviolet Fluorescence. ASTM D130 Corrosion - Copper Strip
Biodiesel-Cu 1a Corrosion 50.degree. C. (122.degree. F.)/3 hr ASTM
Cloud Point Cloud Point 6 .degree. C. D2500 ASTM Micro Carbon
Residue Average Micro <0.10 Wt % D4530 Method Carbon Residue
ASTM D664 Acid Number of Petroleum Procedure Used A Products by
Potentiometric Acid Number 0.20 mg Titration KOH/g ASTM
Determination of Free and Free Glycerin <0.005 Wt % D6584 Total
Glycerin in B-100 Total Glycerin 0.123 Wt % Biodiesel Methyl Esters
By Gas Chromatography ASTM Additive Elements in Phosphorus 0.000200
Wt % D4951 Lubricating Oils by ICP-AES ASTM Distillation of
Petroleum IBP 248 .degree. C. D1160 Products at Reduced Pressure
AET @ 5% 336 .degree. C. Recovery AET @ 10% 338 .degree. C.
Recovery AET @ 20% 339 .degree. C. Recovery AET @ 30% 340 .degree.
C. Recovery AET @ 40% 342 .degree. C. Recovery AET @ 50% 344
.degree. C. Recovery AET @ 60% 345 .degree. C. Recovery AET @ 70%
347 .degree. C. Recovery AET @ 80% 349 .degree. C. Recovery AET @
90% 351 .degree. C. Recovery AET @ 95% 353 .degree. C. Recovery FBP
362 .degree. C. % Recovered 98.5 % % Loss 1.5 % % Residue 0.0 %
Cold Trap Volume 0.0 ml IBP 248 .degree. C. EN 14112 Determination
of Oxidation Oxidation Stability >12 hr Stability (Accelerated
Operating Temp 110 .degree. C. Oxidation Test) (usually 110 deg C)
ASTM Density of Liquids by Digital API Gravity @ 60.degree. F. 29.5
.degree. API D4052 Density Meter ASTM D6890 Determination of
Ignition Derived Cetane >61.0 Delay (ID) and Derived Number
(DCN) Cetane Number (DCN)
[0340] The Cold Soak Filterability by the ASTM D6751 A1 method of
the biodiesel produced was 120 seconds for a volume of 300 ml. This
test involves filtration of 300 ml of B100, chilled to 40.degree.
F. for 16 hours, allowed to warm to room temp, and filtered under
vacuum using 0.7 micron glass fiber filter with stainless steel
support. Oils of the invention can be transesterified to generate
biodiesel with a cold soak time of less than 120 seconds, less than
100 seconds, and less than 90 seconds.
[0341] B. Production of Renewable Diesel
[0342] Degummed oil from Prototheca moriformis UTEX 1435, produced
according to the methods described above and having the same lipid
profile as the oil used to make biodiesel above, was subjected to
transesterification to produce renewable diesel.
[0343] The oil was first hydrotreated to remove oxygen and the
glycerol backbone, yielding n-paraffins. The n-parrafins were then
subjected to cracking and isomerization. A chromatogram of the
material is shown in FIG. 1. The material was then subjected to
cold filtration, which removed about 5% of the C18 material.
Following the cold filtration the total volume material was cut to
flash point and evaluated for flash point, ASTM D-86 distillation
distribution, cloud point and viscosity. Flash point was 63.degree.
C.; viscosity was 2.86 cSt (centistokes); cloud point was 4.degree.
C. ASTM D86 distillation values are shown in Table 7:
TABLE-US-00008 TABLE 7 Readings in .degree. C.: Volume Temperature
IBP 173 5 217.4 10 242.1 15 255.8 20 265.6 30 277.3 40 283.5 50
286.6 60 289.4 70 290.9 80 294.3 90 300 95 307.7 FBP 331.5
[0344] The T10-T90 of the material produced was 57.9.degree. C.
Methods of hydrotreating, isomerization, and other covalent
modification of oils disclosed herein, as well as methods of
distillation and fractionation (such as cold filtration) disclosed
herein, can be employed to generate renewable diesel compositions
with other T10-T90 ranges, such as 20, 25, 30, 35, 40, 45, 50, 60
and 65.degree. C. using triglyceride oils produced according to the
methods disclosed herein.
[0345] The T10 of the material produced was 242.1.degree. C.
Methods of hydrotreating, isomerization, and other covalent
modification of oils disclosed herein, as well as methods of
distillation and fractionation (such as cold filtration) disclosed
herein, can be employed to generate renewable diesel compositions
with other T10 values, such as T10 between 180 and 295, between 190
and 270, between 210 and 250, between 225 and 245, and at least
290.
[0346] The T90 of the material produced was 300.degree. C. Methods
of hydrotreating, isomerization, and other covalent modification of
oils disclosed herein, as well as methods of distillation and
fractionation (such as cold filtration) disclosed herein can be
employed to generate renewable diesel compositions with other T90
values, such as T90 between 280 and 380, between 290 and 360,
between 300 and 350, between 310 and 340, and at least 290.
[0347] The FBP of the material produced was 300.degree. C. Methods
of hydrotreating, isomerization, and other covalent modification of
oils disclosed herein, as well as methods of distillation and
fractionation (such as cold filtration) disclosed herein, can be
employed to generate renewable diesel compositions with other FBP
values, such as FBP between 290 and 400, between 300 and 385,
between 310 and 370, between 315 and 360, and at least 300.
Example 4
Glycerolipid Profile of Prototheca Strains
[0348] Five Prototheca strains were cultivated in media with 2%
glucose and grown for 7 days at 28.degree. C. with agitation (200
rpm) in a 6-well plate. Lipid profiles were determined using
standard HPLC methods. The lipid profile for a particular strain
did not change significantly when grown in different culture media.
The results are shown in Table 8, below.
TABLE-US-00009 TABLE 8 Glycerolipid profile of Prototheca strains.
Origin Species C:12:0 C:13:0 C:14:0 C:16:0 C16:1 C:18:0 C:18:1
C:18:2 C:18:3 UTEX Prototheca 0% 0% 0% 15% 0% 0% 63% 22% 0% 327
stagnora UTEX Prototheca 0% 0% 0% 27% 0% 3% 57% 13% 0% 1439
moriformis UTEX Prototheca 0% 0% 1% 28% 1% 3% 54% 12% 1% 1441
moriformis UTEX Prototheca 0% 0% 1% 26% 0% 3% 55% 12% 2% 1435
moriformis UTEX Prototheca 0% 0% 0% 25% 0% 2% 57% 12% 3% 1437
moriformis
[0349] Biomass from UTEX 1435 was subjected to hexane extraction.
The extracted oil contained very little coloration.
[0350] Although this invention has been described in connection
with specific embodiments thereof, it will be understood that it is
capable of further modifications. This application is intended to
cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth.
[0351] All references cited herein, including patents, patent
applications, GenBank sequences, and publications are hereby
incorporated by reference in their entireties, whether previously
incorporated or not. The publications mentioned therein are cited
for purpose of describing and disclosing reagents, methodologies
and concepts that may be used in connection with the present
invention. Nothing herein is to be construed as an admission that
these references are prior art in relation to the inventions
described therein. In particular, the following patent applications
are hereby incorporated by reference in their entireties for all
purposes: U.S. Provisional Application No. 61/043,620 filed Apr. 9,
2008, entitled "Direct Chemical Modification of Microbial Biomass";
U.S. Provisional Application No. 61/074,610, filed Jun. 20, 2008,
entitled "Soaps and Cosmetic Products Produced from Oil-Bearing
Microbial Biomass and Oils"; International publication number WO
2008/151149; U.S. Patent Application No. 61/118,590, filed Nov. 28,
2008, entitled "Production of Oil in Microorganisms"; U.S.
Provisional Patent Application No. 61/118,994, filed Dec. 1, 2008,
entitled "Production of Oil in Microorganisms"; U.S. Provisional
Patent Application No. 61/174,357, filed Apr. 3, 2009, entitled
"Production of Oil in Microorganisms"; U.S. Provisional Patent
Application No. 61/219,525, filed Jun. 23, 2009, entitled
"Production of Oil in Microorganisms"; U.S. patent application Ser.
No. 12/628,149, filed Nov. 30, 2009, entitled "Renewable Chemical
Production from Novel Fatty Acid Feedstocks", and International
Application No. PCT/US2009/66142, filed Nov. 30, 2009, entitled
"Production of Tailored Oils in Heterotrophic Microorganisms".
Sequence CWU 1
1
11122DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1tgttgaagaa tgagccggcg ac 22220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2cagtgagcta ttacgcactc 203541DNAPrototheca kruegani 3tgttgaagaa
tgagccggcg agttaaaaag agtggcatgg ttaaagaaaa tactctggag 60ccatagcgaa
agcaagttta gtaagcttag gtcattcttt ttagacccga aaccgagtga
120tctacccatg atcagggtga agtgttagta aaataacatg gaggcccgaa
ccgactaatg 180ttgaaaaatt agcggatgaa ttgtgggtag gggcgaaaaa
ccaatcgaac tcggagttag 240ctggttctcc ccgaaatgcg tttaggcgca
gcagtagcag tacaaataga ggggtaaagc 300actgtttctt ttgtgggctt
cgaaagttgt acctcaaagt ggcaaactct gaatactcta 360tttagatatc
tactagtgag accttggggg ataagctcct tggtcaaaag ggaaacagcc
420cagatcacca gttaaggccc caaaatgaaa atgatagtga ctaaggatgt
gggtatgtca 480aaacctccag caggttagct tagaagcagc aatcctttca
agagtgcgta atagctcact 540g 5414573DNAPrototheca wickerhamii
4tgttgaagaa tgagccggcg acttaaaata aatggcaggc taagagattt aataactcga
60aacctaagcg aaagcaagtc ttaatagggc gtcaatttaa caaaacttta aataaattat
120aaagtcattt attttagacc cgaacctgag tgatctaacc atggtcagga
tgaaacttgg 180gtgacaccaa gtggaagtcc gaaccgaccg atgttgaaaa
atcggcggat gaactgtggt 240tagtggtgaa ataccagtcg aactcagagc
tagctggttc tccccgaaat gcgttgaggc 300gcagcaatat atctcgtcta
tctaggggta aagcactgtt tcggtgcggg ctatgaaaat 360ggtaccaaat
cgtggcaaac tctgaatact agaaatgacg atatattagt gagactatgg
420gggataagct ccatagtcga gagggaaaca gcccagacca ccagttaagg
ccccaaaatg 480ataatgaagt ggtaaaggag gtgaaaatgc aaatacaacc
aggaggttgg cttagaagca 540gccatccttt aaagagtgcg taatagctca ctg
5735541DNAPrototheca stagnora 5tgttgaagaa tgagccggcg agttaaaaaa
aatggcatgg ttaaagatat ttctctgaag 60ccatagcgaa agcaagtttt acaagctata
gtcatttttt ttagacccga aaccgagtga 120tctacccatg atcagggtga
agtgttggtc aaataacatg gaggcccgaa ccgactaatg 180gtgaaaaatt
agcggatgaa ttgtgggtag gggcgaaaaa ccaatcgaac tcggagttag
240ctggttctcc ccgaaatgcg tttaggcgca gcagtagcaa cacaaataga
ggggtaaagc 300actgtttctt ttgtgggctt cgaaagttgt acctcaaagt
ggcaaactct gaatactcta 360tttagatatc tactagtgag accttggggg
ataagctcct tggtcaaaag ggaaacagcc 420cagatcacca gttaaggccc
caaaatgaaa atgatagtga ctaaggacgt gagtatgtca 480aaacctccag
caggttagct tagaagcagc aatcctttca agagtgcgta atagctcact 540g
5416541DNAPrototheca moriformis 6tgttgaagaa tgagccggcg agttaaaaag
agtggcatgg ttaaagataa ttctctggag 60ccatagcgaa agcaagttta acaagctaaa
gtcacccttt ttagacccga aaccgagtga 120tctacccatg atcagggtga
agtgttggta aaataacatg gaggcccgaa ccgactaatg 180gtgaaaaatt
agcggatgaa ttgtgggtag gggcgaaaaa ccaatcgaac tcggagttag
240ctggttctcc ccgaaatgcg tttaggcgca gcagtagcaa cacaaataga
ggggtaaagc 300actgtttctt ttgtgggctt cgaaagttgt acctcaaagt
ggcaaactct gaatactcta 360tttagatatc tactagtgag accttggggg
ataagctcct tggtcaaaag ggaaacagcc 420cagatcacca gttaaggccc
caaaatgaaa atgatagtga ctaaggatgt gggtatgtta 480aaacctccag
caggttagct tagaagcagc aatcctttca agagtgcgta atagctcact 540g
5417573DNAPrototheca moriformis 7tgttgaagaa tgagccggcg acttaaaata
aatggcaggc taagagaatt aataactcga 60aacctaagcg aaagcaagtc ttaatagggc
gctaatttaa caaaacatta aataaaatct 120aaagtcattt attttagacc
cgaacctgag tgatctaacc atggtcagga tgaaacttgg 180gtgacaccaa
gtggaagtcc gaaccgaccg atgttgaaaa atcggcggat gaactgtggt
240tagtggtgaa ataccagtcg aactcagagc tagctggttc tccccgaaat
gcgttgaggc 300gcagcaatat atctcgtcta tctaggggta aagcactgtt
tcggtgcggg ctatgaaaat 360ggtaccaaat cgtggcaaac tctgaatact
agaaatgacg atatattagt gagactatgg 420gggataagct ccatagtcga
gagggaaaca gcccagacca ccagttaagg ccccaaaatg 480ataatgaagt
ggtaaaggag gtgaaaatgc aaatacaacc aggaggttgg cttagaagca
540gccatccttt aaagagtgcg taatagctca ctg 5738573DNAPrototheca
wickerhamii 8tgttgaagaa tgagccgtcg acttaaaata aatggcaggc taagagaatt
aataactcga 60aacctaagcg aaagcaagtc ttaatagggc gctaatttaa caaaacatta
aataaaatct 120aaagtcattt attttagacc cgaacctgag tgatctaacc
atggtcagga tgaaacttgg 180gtgacaccaa gtggaagtcc gaaccgaccg
atgttgaaaa atcggcggat gaactgtggt 240tagtggtgaa ataccagtcg
aactcagagc tagctggttc tccccgaaat gcgttgaggc 300gcagcaatat
atctcgtcta tctaggggta aagcactgtt tcggtgcggg ctatgaaaat
360ggtaccaaat cgtggcaaac tctgaatact agaaatgacg atatattagt
gagactatgg 420gggataagct ccatagtcga gagggaaaca gcccagacca
ccagttaagg ccccaaaatg 480ataatgaagt ggtaaaggag gtgaaaatgc
aaatacaacc aggaggttgg cttagaagca 540gccatccttt aaagagtgcg
taatagctca ctg 5739541DNAPrototheca moriformis 9tgttgaagaa
tgagccggcg agttaaaaag agtggcgtgg ttaaagaaaa ttctctggaa 60ccatagcgaa
agcaagttta acaagcttaa gtcacttttt ttagacccga aaccgagtga
120tctacccatg atcagggtga agtgttggta aaataacatg gaggcccgaa
ccgactaatg 180gtgaaaaatt agcggatgaa ttgtgggtag gggcgaaaaa
ccaatcgaac tcggagttag 240ctggttctcc ccgaaatgcg tttaggcgca
gcagtagcaa cacaaataga ggggtaaagc 300actgtttctt ttgtgggctc
cgaaagttgt acctcaaagt ggcaaactct gaatactcta 360tttagatatc
tactagtgag accttggggg ataagctcct tggtcgaaag ggaaacagcc
420cagatcacca gttaaggccc caaaatgaaa atgatagtga ctaaggatgt
gagtatgtca 480aaacctccag caggttagct tagaagcagc aatcctttca
agagtgcgta atagctcact 540g 54110541DNAPrototheca zopfii
10tgttgaagaa tgagccggcg agttaaaaag agtggcatgg ttaaagaaaa ttctctggag
60ccatagcgaa agcaagttta acaagcttaa gtcacttttt ttagacccga aaccgagtga
120tctacccatg atcagggtga agtgttggta aaataacatg gaggcccgaa
ccgactaatg 180gtgaaaaatt agcggatgaa ttgtgggtag gggcgaaaaa
ccaatcgaac tcggagttag 240ctggttctcc ccgaaatgcg tttaggcgca
gcagtagcaa cacaaataga ggggtaaagc 300actgtttctt tcgtgggctt
cgaaagttgt acctcaaagt ggcaaactct gaatactcta 360tttagatatc
tactagtgag accttggggg ataagctcct tggtcaaaag ggaaacagcc
420cagatcacca gttaaggccc caaaatgaaa atgatagtga ctaaggatgt
gagtatgtca 480aaacctccag caggttagct tagaagcagc aatcctttca
agagtgcgta atagctcact 540g 54111565DNAPrototheca moriformis
11tgttgaagaa tgagccggcg acttagaaaa ggtggcatgg ttaaggaaat attccgaagc
60cgtagcaaaa gcgagtctga atagggcgat aaaatatatt aatatttaga atctagtcat
120tttttctaga cccgaacccg ggtgatctaa ccatgaccag gatgaagctt
gggtgatacc 180aagtgaaggt ccgaaccgac cgatgttgaa aaatcggcgg
atgagttgtg gttagcggtg 240aaataccagt cgaacccgga gctagctggt
tctccccgaa atgcgttgag gcgcagcagt 300acatctagtc tatctagggg
taaagcactg tttcggtgcg ggctgtgaga acggtaccaa 360atcgtggcaa
actctgaata ctagaaatga cgatgtagta gtgagactgt gggggataag
420ctccattgtc aagagggaaa cagcccagac caccagctaa ggccccaaaa
tggtaatgta 480gtgacaaagg aggtgaaaat gcaaatacaa ccaggaggtt
ggcttagaag cagccatcct 540ttaaagagtg cgtaatagct cactg 565
* * * * *
References